JP5069130B2 - Oral formulations for delivery of catecholbutane including NDGA compounds - Google Patents

Description

  The present invention relates to oral formulations for the delivery of catecholbutane including NDGA compounds.

This application claims the benefit of US Provisional Patent Applications Nos. 60 / 647,495 and 60 / 647,648, both filed Jan. 27, 2005, which are hereby incorporated by reference in their entirety. Are incorporated herein. This application is also filed at the same time as this application and is an international patent application identified as agent case number 682714-10WO entitled “Formulation for injection of catecholbutane containing NDGA compounds into animals” The disclosure of which is hereby incorporated by reference in its entirety.

  The present application relates to compositions for the administration of catecholbutane, such as NDGA derivatives, to animals such as humans for the treatment of diseases such as cancer or other proliferative or inflammatory diseases, metabolic diseases or neurodegenerative diseases. It relates to things and methods.

  Catecholbutanes such as nordihydroguaiaretic acid (NDGA) are being tested for therapeutic use in laboratory animals by intratumoral injection or topical application of such compounds. For example, Jordan et al. Described the effect of NDGA on human breast cancer MX-1 implanted subcutaneously in mice on day 0 in US Pat. No. 5,008,294 (Example 2). Tumor-bearing mice were injected with various doses of NDGA on day 1 with a single intratumoral injection. In another experiment, Jordan et al. Injected human breast adenocarcinoma MX-1 intradermally into mice and treated the animals with topical application of NDGA 23 days later (Example 7).

Huang et al., In US Pat. No. 6,214,874, describes the testing of certain derivatives of NDGA (ie, NDGA derivatives). In one experiment, mice were injected with an immortalized mouse cell line, C3 cells, and treated by intratumoral injection of M ₄ N alone or in combination with G ₄ N, both derivatives of NDGA.

  It would be desirable if oral formulations of these anti-cancer compounds were discovered that would allow these easier administrations, particularly self-administration without the need for hospitalization. The present invention provides these desirable advantages.

  Accordingly, one object of the present invention is to provide one or more new oral formulations of catecholbutane and / or NDGA compounds, such as NDGA derivatives, which are intended for subjects in need of treatment. For the treatment of diseases by oral administration of such formulations.

  Another object of the present invention is to provide a formulation as described above that is safe and has fewer adverse side effects when administered to animals, including humans.

  Another object of the present invention is to provide a formulation as one or more of the above that is stable for a commercially reasonable period of time.

  Another object of the present invention is to provide a formulation as one or more of the above having a commercially reasonable half-life in circulation for administration to an animal.

  In accordance with one or more of the objects of the invention described above, embodiments of the invention illustrated below are provided.

  A composition for oral administration to an animal comprising an active pharmaceutical ingredient and a pharmaceutically acceptable carrier, wherein the active pharmaceutical ingredient comprises catecholbutane, and the carrier is (a) a water-soluble organic solvent, However, when the water-soluble organic solvent is propylene glycol, the propylene glycol is in the absence of white petrolatum, in the absence of xanthan gum (also known as xantham gum xanthum gum), and at least one of glycerin or glycine. If present and the water-soluble organic solvent is polyethylene glycol, the polyethylene glycol is present in the absence of ascorbic acid or butylated hydroxytoluene (BHT) and the polyethylene glycol is polyethylene glycol 400 If the polyethylene glycol 400 is a polyethylene glycol Present in the absence of Recall 8000; (b) cyclodextrin; (c) ionic, non-ionic or amphiphilic surfactant, but not if the surfactant is a non-ionic surfactant An ionic surfactant is present in the absence of xanthan gum; (d) modified cellulose; (e) a water-insoluble lipid, but if the water-insoluble lipid is castor oil, the castor oil is absent of beeswax or carnauba wax A composition comprising at least one of a solubilizer and an excipient selected from the group consisting of: an underneath; and any combination of carriers (a)-(e).

  A method of treating a disease in a subject comprising: (a) providing a composition of the present invention; and (b) orally administering said composition to said subject, said composition comprising an effective amount of said A method comprising an active pharmaceutical ingredient.

  A kit for the treatment of a disease comprising the composition of the invention and instructions for its use.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. However, the invention is not limited to the precise arrangements and instrumentalities shown.

  The drawings are as follows.

FIG. 1 includes the results of a cell proliferation test performed on the C-33A cell line and the HeLa cell line after M ₄ N treatment, including FIGS. 1A and 1B. 1A is an illustration of the ratio of the number of cells present after M _{4 N} treatment exceeds the number of existing cells in the absence of M 4 _N treated graph, where M _{4 N} is DMSO formulation Medium was provided in amounts ranging from 0 μM to 80 μM. Figure 1B, is shown graphically the ratio of the number of cells present after M _{4 N} treatment exceeds the number of cells present in the M _{4 N} treatment absence, where M _{4 N} is, HP-beta -Offered in amounts ranging from 0 [mu] M to 80 [mu] M in CD / PEG formulation ("CPE" formulation).

FIG. 2 includes FIGS. 2A and 2B, and in the absence or presence of various concentrations of M ₄ N in the DMSO (FIG. 2A) formulation or in the HP-β-CD / PEG formulation (FIG. 2B). The measurement of the cell death based on the ratio of the dead cell regarding C-33A cell and HeLa cell is shown with the graph. The M ₄ N concentration was in the range of 0 μM to 80 μM.

FIG. 3 shows 0.5 hours, 1 hour, 2 hours and 3 hours after oral administration of a single 500 mg / kg amount of M ₄ N in various liquid solubilizers and / or excipients (“carriers”). it is a histogram showing the absorption of _{M 4} N in Sprague Dawley rats at the time of. M ₄ N was present at a concentration of about 60 mg / mL in all carriers. The carrier is (a) HP-β-CD (500 mg / mL) + HPMC (5 mg / mL); (b) HP-β-CD (500 mg / mL) + CMC (5 mg / mL); (c) TPGS (200 mg / mL) ); (D) TPGS (100 mg / mL) + PEG 400 (50% v / v); (e) Tween (registered trademark: hereinafter omitted, “Tween” is a registered trademark) 20; (f) PEG 400 (50% v / v) + Tween 20 (50% v / v); (g) PEG 400 (33% v / v) + Tween 20 (33% v / v) + peppermint oil (33%); (h) Peppermint oil (50%) + PEG 400 (50% v / v); (h) Peppermint oil (50%) + Tween 20 (50% v / v); (i) Peppermint oil (50%) + sesame oil (50%) It encompasses.

FIG. 4 shows the powder form or various solid carriers: (a) M ₄ N powder; (b) lyophilized HP-β-CD (81%) + M ₄ N (185 mg / g); (c) TPGS (20%) + M ₄ N (133 mg / g); (d) soybean oil (95%) + beeswax (5%) + M ₄ N (200 mg / g); and (d) olive oil (95%) + beeswax (5 %) + M ₄ N (200 mg / g) of M ₄ N at a single dose of 100 mg / kg 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours and 8 hours The graph shows M ₄ N absorption by beagle dogs at the time point.

FIG. 5 includes FIGS. 5A and 5B, and includes 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and a single 100 mg / kg dose after oral administration. FIG. 3 is a graphical representation of the absorption of non-micronized M ₄ N in glycerol monooleic acid by a beagle dog at 36 hours. FIG. 5A is on a non-logarithmic scale. FIG. 5B is on a logarithmic scale.

FIG. 6 includes FIGS. 6A and 6B, and includes 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and a single 100 mg / kg oral dose. ₃ is a graphical representation of the absorption of micronized M ₄ N in glycerol monooleic acid by a beagle dog at 36 hours. FIG. 6A is on a non-logarithmic scale. FIG. 6B is on a logarithmic scale.

FIG. 7 includes FIGS. 7A and 7B, and 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours after oral administration of a single 75 mg / kg oral dose. 2 graphically shows the serum concentration of M ₄ N in various carriers at various concentrations by male beagle dogs at time, 24 hours and 36 hours. The abbreviations for the carriers used, the concentration of M ₄ N administered, and the designation of whether it was administered to fasted or fed dogs are most clearly shown in FIG. 7B. FIG. 7A shows data on non-logarithmic scales. FIG. 7B shows data on a logarithmic scale.

FIG. 8 includes FIGS. 8A and 8B, and 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours after oral administration of a single 75 mg / kg oral dose. 2 graphically illustrates the serum concentration of M ₄ N in various carriers at various concentrations by female beagle dogs at time, 24 hours and 36 hours. The abbreviation of the carrier used, the concentration of M ₄ N administered, and the designation of whether it was administered to a fasted or fed dog is most clearly shown in FIG. 8B. FIG. 8A shows data on a non-logarithmic scale. FIG. 8B shows data on a logarithmic scale.

Detailed description of the invention The present invention relates to proliferative diseases such as cancer and psoriasis, hypertension, obesity, diabetes, types I and II, diseases of the central nervous system or neurodegenerative disorders, including but not limited to pain, Alzheimer's disease, stroke And inflammatory diseases, premalignant neoplasia or dysplasia, infections such as viral infections such as HIV, HTLV, HPV, HSV, HBV, EBV, varicella zoster, adenovirus, parvovirus, JC virus, etc. Novel compositions, kits and methods for the treatment of diseases comprising The present invention relates to proliferative diseases such as cancer and psoriasis, hypertension, obesity, type I and type II diabetes, central nervous system diseases or neurodegenerative diseases such as pain, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Dementia, stroke and inflammatory diseases, premalignant neoplasia or dysplasia, infections such as viral infections, including but not limited to human immunodeficiency virus ("HIV"), human T cell leukemia virus ("HTLV") ), Human papillomavirus ("HPV"), herpes simplex virus ("HSV"), hepatitis B virus ("HBV"), Epstein-Barr virus ("EBV"), varicella-zoster, adenovirus, parvovirus, Jacob Novel compositions, kits and methods for the treatment of diseases including those caused by Creutzfeldt virus ("JC virus") To provide.

The present invention provides novel compositions containing catecholbutane, including NDGA derivatives, eg, NDGA compounds such as M ₄ N, dissolved in certain pharmaceutically acceptable solubilizers. In combination with other diluents, excipients and the like (collectively referred to as “carriers”) constitutes a formulation that is suitable for administration to a subject, such as a human, for the treatment of a disease. Such formulations are suitable for oral administration. Suitable pharmaceutically acceptable carriers are (a) water soluble organic solvents other than DMSO, such as polyethylene glycol ("PEG"), such as PEG 300, PEG 400 or PEG 400 monolauric acid, propylene glycol ("PG"). , Polyvinylpyrrolidone (“PVP”), ethanol, benzyl alcohol or dimethylacetamide; (b) cyclodextrin or modified cyclodextrins such as hydroxypropyl-β-cyclodextrin (“HP-β-CD”) or sulfobutyl ether β-cyclo Dextrin (“SBE-β-CD”); (c) ionic, non-ionic or amphiphilic surfactants such as polyoxyethylene sorbitan monolauric acid (also known as polysorbate) which is a non-ionic surfactant An illustration) Polysorbate 20 and polysorbate 80, commercially available as Tween 20 or Tween 80, d-alpha-tocopheryl polyethylene glycol 1000 succinic acid ("TPGS"), glycerol monooleic acid (also known as glyceryl monooleic acid), Esterified fatty acid or Cremophor (registered trademark: hereinafter, “Cremophor” is a registered trademark). The reaction product between ethylene oxide and castor oil in a molar ratio of 35: 1 marketed as EL; (d ) Modified cellulose, such as ethylcellulose (“EC”), hydroxylpropylmethylcellulose (“HPMC”), methylcellulose (“MC”) or carboxymethylcellulose (“CMC”); and (e) water-insoluble lipids, eg If the wax, oil or fat emulsion, for example Intralipid: include (R hereinafter omitted "Intralipid" as a trademark.) At least one member selected from. Preferably, when PG is used, it is used in the absence of white petrolatum, in the absence of xanthan gum, and in the absence of at least one of glycerin or glycine. Preferably, if PEG is used, it is used in the absence of ascorbic acid or BHT; if a nonionic surfactant is used, it is used in the absence of xanthan gum; and the oil is castor If oil, it is in the absence of beeswax or carnauba wax.

  In one embodiment of the invention, the compounds described herein are dissolved in various carriers or dissolved and diluted to form oral liquid compositions for administration to animals. For example, in one aspect of embodiments, the active pharmaceutical ingredient (“API”) compound of the invention is in a water-soluble organic solvent, such as PEG 300, PEG 400 or PEG 400 monolauric acid (“PEG compound”), Or it dissolves in PG. In another embodiment, the compounds described herein are dissolved in a modified cyclodextrin, such as HP-β-CD or SBE-β-CD. In yet another embodiment, the compounds of the invention are solubilized and / or diluted in a combined formulation containing a PEG compound and HP-β-CD. In another embodiment, the compounds described herein are dissolved in a modified cellulose, such as HPMC, CMC or EC. In yet another embodiment, the compound described herein is a modified combination containing a modified cyclodextrin and a modified cellulose, such as both HP-β-CD and HPMC or HP-β-CD and CMC. Dissolve in.

  In yet another embodiment, the compounds described herein are soluble in ionic, non-ionic or amphiphilic surfactants such as Tween 20, Tween 80, TPGS or esterified fatty acids. For example, the compounds of the present invention can be dissolved in TPGS alone, or Tween 20 alone, or TPGS and PEG 400, or a combination of Tween 20 and PEG 400.

  In another embodiment, the compounds of the invention are soluble in water insoluble lipids such as waxes, fat emulsions such as Intralipid, or oil. For example, the compounds of the present invention can be dissolved in peppermint oil alone or in a combination of peppermint oil and Tween 20 and PEG 400, or peppermint oil and PEG 400, or peppermint oil and Tween 20, or peppermint oil and sesame oil.

  Of course, ethylcellulose may replace HPMC or CMC in the above example, or may be added instead; PEG 300 or PEG 400 monolauric acid may replace or be added to PEG 400 in the above example; Tween 80 may replace or be added to Tween 20 in the above example; and other oils such as corn oil, olive oil, soybean oil, mineral oil or glycerol may be replaced with peppermint oil or sesame oil in the above example Or may be added.

  In addition, heating may be applied, for example, to achieve dissolution of the compounds described herein by heating to a temperature of about 30 ° C. to about 90 ° C. during the formulation of these compositions, or A homogeneously dispersed suspension of the compounds herein may be obtained.

  In yet another embodiment, the API compounds of the invention are administered orally as a solid without any carrier or using a carrier. In one embodiment, the compounds described herein are first dissolved in a liquid carrier as in the examples above and then made into a solid composition for administration as an oral composition. For example, the compounds of the invention are dissolved in a modified cyclodextrin such as HP-β-CD and the composition is lyophilized to a powder suitable for oral administration.

  In another embodiment, the compounds of the invention are dissolved or suspended in a TPGS solution with appropriate heating to obtain a uniformly dispersed solution or suspension. Upon cooling, the composition becomes creamy and suitable for oral administration.

  In yet another embodiment, the compound of the invention is dissolved in oil and beeswax is added to produce a waxy solid composition.

  In yet another embodiment, the compounds described herein are solubilized in a modified cellulose such as EC. Modified cellulose, such as ethylcellulose, can be diluted in ethanol ("EtOH") prior to use.

  The present invention also provides water insoluble lipids as solubilizers for the compounds of the present invention. Water insoluble lipids include, for example, oils as well as mixed fat emulsion compositions such as Intralipid (Pharmacia & Upjohn, now Pfizer) used according to manufacturer's recommendations. For example, it is recommended that adult doses should not exceed 2 g fat / kg body weight / day (Intralipid 10%, 20% and 30%, 20 mL, 10 mL and 6.7 mL / kg, respectively). Intralipid 10% is said to contain 100 g of purified soybean oil, 12 g of purified egg phospholipid, 22 g of anhydrous glycerol, and a total volume of 1000 mL with water for injection in 1000 mL. The pH is adjusted to approximately pH 8 using sodium hydroxide. Intralipid 20% contains 200 g of refined soybean oil, 12 g of purified egg phospholipid, 22 g of anhydrous glycerol, and 1000 L in water for injection in 1000 mL. The pH is adjusted to approximately pH 8 using sodium hydroxide. Intralipid 30% contains 300 g of purified soybean oil, 12 g of purified egg phospholipid, 16.7 g of anhydrous glycerol, and a total volume of 1000 mL with water for injection in 1000 mL. The pH is adjusted to approximately pH 7.5 using sodium hydroxide. Intralipid products should be stored at a controlled room temperature below 25 ° C. and not frozen.

  In general, in the preparation of oral formulations, the compounds described herein are first solubilized and then other excipients are added to produce a composition with a higher degree of stability. Unstable formulations are not desirable. Unstable liquid formulations often form crystalline precipitates or biphasic solutions. Unstable solid formulations often have a granular, clumpy appearance and often contain a flowable liquid. The optimal solid formulation has a smooth and homogeneous appearance and a narrow melting temperature range. In general, the ratio of excipients in the formulation affects stability. Substances that are too small in curability, such as beeswax, make the formulation too fluid.

Thus, in general, for the liquid formulations of the present invention, the excipient used must be a good solvent for the API or a compound described herein, eg, M ₄ N. In other words, the excipient must be able to dissolve the API without overheating. Excipients must also be compatible with each other independently of the API so that they can form stable solutions, suspensions, or emulsions. Furthermore, in general, in the case of the solid formulations of the present invention, the excipient used should also be a good solvent for the API to avoid lumps and non-uniform formulations. In order to avoid undesirably high fluidity or non-homogeneous textured solid formulations, the excipients are compatible with each other to form a smooth, uniform solid even in the absence of the API. There must be.

  The present invention also provides “placebo” formulations that are comparable to, but do not contain, APIs. These placebo formulations are useful for examining the suitability of various ingredients or excipients in the formulation.

  Terms used herein have their usual lexical meaning and are used as known in the art unless otherwise specified. In particular, the present invention can be better understood with reference to the following definitions, which are used in conjunction with other terms defined elsewhere in this specification.

  The term “about” as used herein when referring to a concentration or dose means a specific number from ± 10% to 20%.

  Reference to the term “active pharmaceutical ingredient”, “API” or “compound” as used herein refers to a catecholbutane of formula I or an NDGA compound present in the pharmaceutical composition described herein. , For example, means one or more of NDGA derivatives.

  The term “alkylenedioxy” as used herein refers to methylene or substituted methylenedioxy or ethylene or substituted ethylenedioxy.

  An “unsubstituted or substituted amino acid residue or salt thereof” when referring to one of the —R groups in Formula I or Formula II is used herein to refer to an aromatic group in Formula I or Formula II. Means an amino acid having a reduced “H” or substituted amino acid at its C-terminus due to its own ligation, wherein the amino acid or substituted amino acid includes, but is not limited to, for example, alanine, Arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5-hydroxylysine, 4-hydroxyproline, thyroxine , 3-methylhistidine, ε-N-methyllysine, ε-N, N, N-trimethyllysine, aminoadipic acid, γ-carboxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine, N-methylarginine, N-acetyllysine and N, N-dimethyl substituted amino acids or any of the salts mentioned above, such as chloride Includes salt.

  A “buffer” suitable for use in the present invention is any conventional buffer known in the art and includes, for example, Tris, phosphate, imidazole and bicarbonate.

  “Carrier” as used herein refers to a non-toxic solid, semi-solid or liquid filler, diluent, vehicle, excipient, solubilizer, encapsulating material or any The conventional type of pharmaceutical adjuncts and includes all components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional substances such as wettable or emulsifying agents, or pH buffering agents. Other materials such as antioxidants, wetting agents, viscosity stabilizers and similar materials may be added as needed.

  “Catecholbutane” as used herein means a compound of formula I:

ここで、式中、Ｒ_１及びＲ_２は各々独立して−Ｈ、低級アルキル、低級アシル、アルキレンを示すか；又は−Ｒ_１Ｏ及び−Ｒ_２Ｏは各々独立して未置換又は置換されたアミノ酸残基又はその塩を示し；Ｒ_３、Ｒ_４、Ｒ_５、Ｒ_６、Ｒ_１０、Ｒ_１１、Ｒ_１２及びＲ_１３は各々独立して−Ｈ又は低級アルキルを示し；そしてＲ_７、Ｒ_８及びＲ_９は各々独立して−Ｈ、−ＯＨ、低級アルコキシ、低級アシルオキシ、未置換又は置換されたアミノ酸残基又はその塩を示すか、又は何れかの２つの隣接する基は一緒になってアルキレンジオキシであってよい。

Here, in the formula, R ₁ and R ₂ each independently represent —H, lower alkyl, lower acyl, alkylene; or —R ₁ O and —R ₂ O are each independently unsubstituted or substituted. R ₃ , R ₄ , R ₅ , R ₆ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ each independently represent —H or lower alkyl; and R ₇ , R ₈ and R ₉ each independently represent —H, —OH, lower alkoxy, lower acyloxy, an unsubstituted or substituted amino acid residue or a salt thereof, or any two adjacent groups together. And may be alkylenedioxy.

  “Cyclodextrin” as used herein means unmodified cyclodextrin or modified cyclodextrin, and includes, but is not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and modifications added thereto. Any modified cyclodextrin such as HP-β-CD or SBE-β-CD. Cyclodextrins typically have 6 (α-cyclodextrin), 7 (β-cyclodextrin), 8 (γ-cyclodextrin) sugars, up to 3 substitutions per saccharide, and therefore 0-24 Primary substitution is possible (primary substitution is defined as a substitution directly linked to the cyclodextrin ring). The modified or unmodified cyclodextrins used in the present invention may have either appropriate amounts and positions of primary substitutions or other modifications.

  The “derivative” of NDGA means “NDGA derivative” in the present specification (described later).

  The term “disease” as used herein encompasses any disease, condition, infection, syndrome or disorder for which application of a composition of the invention results in a therapeutic effect. Such “diseases” include, but are not limited to, for example, cancer, psoriasis and other proliferative diseases, inflammatory disorders such as rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, atherosclerosis, Chronic obstructive pulmonary disease (COPD), hypertension, obesity, diabetes, pain, stroke and / or other neurological disorders or neurodegenerative diseases or conditions such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, muscle atrophy Includes lateral sclerosis ("ALS") and premalignant conditions such as intraepithelial neoplasia or dysplasia, and infectious diseases.

“G ₄ N” or “tetra-N, N-dimethylglycinyl NDGA” or “tetradimethylglycinyl NDGA” as used herein refers to R ₁₄ , R ₁₅ , either in solid form or in solution. R ₁₆ and _{R 17} are each independently _{-O (C = O) CH 2} N (CH 3) 2 or ^{_{-O (C = O) CH 2}} N + (CH 3) 2 · Cl - indicates; and R NDGA derivatives of formula II such that ₁₈ and R ₁₉ each represent —CH ₃ .

“Lower acyl” refers herein to C ₁ -C ₆ acyl, preferably C ₁ -C ₃ acyl.

“Lower alkyl” as used herein means C ₁ -C ₆ alkyl, preferably C ₁ -C ₃ alkyl.

“M ₄ N” or “tetra-O-methyl NDGA” as used herein means that R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ each independently represents —OCH ₃ , and R ₁₈ and R NDGA derivatives of formula II, wherein ₁₉ each represents —CH ₃ .

  “Modified cellulose” as used herein refers to cellulose, such as EC, HPMC, CMC, and MC, that contains one or more modifications to the cellulose molecule.

  “NDGA” as used herein means nordihydroguaiaretic acid and has the formula:

  By “NDGA compound” is meant herein one or more of any of NDGA and / or NDGA derivatives, either generically or generically.

  "NDGA derivative" means herein a derivative of NDGA having the following formula II:

ここで、式中、Ｒ_１４、Ｒ_１５、Ｒ_１６及びＲ_１７は、各々独立して−ＯＨ、低級アルコキシ、低級アシルオキシ、又は未置換又は置換されたアミノ酸残基又は製薬上許容しうるその塩を示し；そしてＲ_１８及びＲ_１９は、各々独立して−Ｈ又は低級アルキルを示し、ここでＲ_１４、Ｒ_１５、Ｒ_１６及びＲ_１７は、同時に−ＯＨではない。即ち、用語は、ＮＤＧＡのメチル化誘導体、例えばテトラ−Ｏ−メチルＮＤＧＡ（Ｍ_４Ｎ）、トリ−Ｏ−メチルＮＤＧＡ（Ｍ_３Ｎ）、ジ−Ｏ−メチルＮＤＧＡ（Ｍ_２Ｎ）及びモノ−Ｏ−メチルＮＤＧＡ（Ｍ_１Ｎ）、を包含する。或いは、ＮＤＧＡ誘導体は、ＮＤＧＡのヒドロキシル又はメチル基の水素の１つ以上が置換されている化合物、例えば、Ｒ_１４、Ｒ_１５、Ｒ_１６及びＲ_１７が、各々独立して低級アルコキシ、低級アシルオキシ、又はアミノ酸又は置換されたアミノ酸又はその塩を示し；そしてＲ_１８及びＲ_１９が、各々独立して−Ｈ又はアルキル、例えば低級アルキルを示すものであってよい。用語は、例えば、Ｒ_１４、Ｒ_１５、Ｒ_１６及びＲ_１７が、各々独立して−ＯＣＨ_３又は−Ｏ（Ｃ＝Ｏ）ＣＨ_３又は二置換アミノ酸残基、例えばＮ，Ｎ−ジメチル置換アミノ酸残基、例えば−Ｏ（Ｃ＝Ｏ）ＣＨ_２Ｎ（ＣＨ_３）_２又は−Ｏ（Ｃ＝Ｏ）ＣＨ_２Ｎ^＋（ＣＨ_３）_２・Ｃｌ⁻を示し；そしてＲ_１８及びＲ_１９が、各々独立して−Ｈ又は低級アルキル、例えば−ＣＨ_３又は−ＣＨ_２ＣＨ_３を示す化合物を包含する。

Here, in the formula, R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are each independently —OH, lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or a pharmaceutically acceptable salt thereof. And R ₁₈ and R ₁₉ each independently represent —H or lower alkyl, wherein R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are not simultaneously —OH. That is, the terms are methylated derivatives of NDGA, such as tetra-O-methyl NDGA (M ₄ N), tri-O-methyl NDGA (M ₃ N), di-O-methyl NDGA (M ₂ N) and mono- O-methyl NDGA (M ₁ N). Alternatively, an NDGA derivative is a compound in which one or more of the hydroxyl or methyl groups of NDGA are substituted, for example, R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are each independently lower alkoxy, lower acyloxy, Or an amino acid or a substituted amino acid or a salt thereof; and R ₁₈ and R ₁₉ may each independently represent —H or alkyl, such as lower alkyl. The terms are, for example, R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ are each independently —OCH ₃ or —O (C═O) CH ₃ or a disubstituted amino acid residue, such as an N, N-dimethyl substituted amino acid Represents a residue, for example —O (C═O) CH ₂ N (CH ₃ ) ₂ or —O (C═O) CH ₂ N ⁺ (CH ₃ ) ₂ .Cl ⁻ ; and R ₁₈ and R ₁₉ are Included are compounds each independently representing —H or lower alkyl, eg, —CH ₃ or —CH ₂ CH ₃ .

  As used herein, “ratio”, “percentage” or sign “%” means the proportion of the indicated ingredient in the composition based on the amount of carrier present in the composition, and By weight / weight (w / w), weight / volume (w / v) or volume / volume (v / v) as indicated for a particular component, all of which are the amount of carrier present in the composition. Based on. That is, different types of carriers may be present in amounts up to 100% as indicated, and this does not preclude the presence of API, the amount as a percentage or specific mg present in the composition. It may be shown as a number or a specific mg / g number present or a specific mg / mL number present, where%, mg / g or mg / mL is based on the total amount of carrier present in the composition. The carrier may be 100% due to the presence of various specific carriers in combination.

  "Pharmaceutically acceptable carrier" as used herein is nontoxic to recipients at the dosages and concentrations employed and is compatible with the other ingredients in the formulation. For example, carriers for formulations containing catecholbutane, NDGA compounds or NDGA derivatives of the present invention preferably do not include oxidizing agents and other compounds known to adversely affect them. Pharmaceutically acceptable carriers include solubilizers. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, glucose, glycerol, saline, ethanol, buffer, Cremaphor EL, phosphate buffered saline, PEG 300, PEG 400, modified cyclodextrins and these Includes all of the above, such as combinations.

  The term “pharmaceutically acceptable excipient” includes solvents, adjuvants or diluents or other ancillary substances such as conventional products in the art, which are readily available to the public and used at the dosage and It is non-toxic to recipients at concentrations and is compatible with other ingredients in the formulation. For example, pharmaceutically acceptable auxiliary substances include pH adjusters and buffers, osmotic pressure adjustors, stabilizers, wetting agents and the like.

  The term “solubilizing agent” as used herein means a composition in which one or more catecholbutane or NDGA, eg, an NDGA derivative, is dissolved. The solubilizer may also be a carrier or a pharmaceutically acceptable carrier.

  The terms “subject”, “host” and “patient” refer to animals, such as, but not limited to, monkeys, humans, cats, dogs, horses, cows, pigs, sheep, goats, mammals to be treated by the compositions of the invention. Used interchangeably herein to refer to pastoral animals, mammal sport animals, and mammal pets.

  As used herein, a “substantially purified” compound when referring to a catecholbutane or NDGA derivative substantially refers to a substance that is not a catecholbutane, NDGA compound or NDGA derivative (collectively “non-NDGA substance”). It does not contain. Substantially free contains at least about 50% non-NDGA material, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, and even more preferably at least about Means containing no 95% non-NDGA material.

  As used herein, the terms “treatment” and “treating” refer to obtaining a desired pharmacological and / or physiological effect. The action may be prophylactic in the sense of completely or partially preventing the condition or disease or its symptoms and / or a partial or complete effect of the condition or disease and / or the condition or disease. It may be therapeutic in the sense of healing. “Treatment” of a subject includes treatment of any disease in, for example, mammals, particularly humans, and (a) the disease occurs in a subject that is susceptible to the disease but has not yet been diagnosed as having it (B) inhibiting the development or progression of the disease, for example, stopping its development; and (c) relieving, alleviating or ameliorating the disease. For example, eliciting disease regression or remission.

  Before further describing the present invention, it is to be understood that the present invention is not limited to the specific embodiments described, but may naturally vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention is limited only by the accompanying claims.

  Where a range of values is presented, each intervening value between the upper and lower limits of the range shall be up to 1 / 10th of the unit of adjustment unless otherwise stated, and within the stated range. Any other stated or intervening numerical value of any of the above shall be included in the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the invention and subject to any specifically excluded ranges within the stated ranges. It becomes. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

  In this specification, the singular forms also include plural forms unless the context clearly dictates otherwise. Thus, for example, “a compound” includes a plurality of such compounds, and reference to “NDGA compound” refers to one or more NDGA compounds, such as NDGA derivatives and equivalents known in the art. Is included.

  All publications mentioned herein, for example, patents, patent applications, and journal articles, are hereby incorporated by reference in their entirety, including references cited therein that are incorporated herein by reference in their entirety. Incorporated into.

  The publications discussed herein are presented solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the dates of publications presented may differ from the actual publication dates that need to be individually confirmed. Citations of references cited within the text of this application are more fully identified in the bibliography preceding the claims.

  The invention described below is presented by way of example only and should not be construed as limiting the invention in any way.

  Preparation of catecholbutane

  The catecholbutane of the present invention can be made by any method known in the art. For example, such compounds can be prepared as described in US Pat. No. 5,008,294.

  Production of NDGA derivatives

  NDGA is a commercial product manufacturer such as Alexis Biochemicals Corp., San Diego, CA, USA (Catalog Number LKT-N5669) or AG Scientific, Inc., San Diego, CA, USA (Catalog Number N1071) or Cayman Chemical Company, Ann Arbor. , MI, USA (Cat. No. 70300).

  The NDGA derivative and its formulation may be prepared by any conventional method in the art. For example, NDGA derivatives are described in US Pat. No. 5,008,294; US Pat. No. 6,291,524; Hwu, JR et al. (1998); or McDonald, McDonald, RW) et al. (2001).

In one embodiment of the invention, NDGA derivative, tetra-O-methyl NDGA, aka meso-1,4-bis (3,4-dimethoxyphenyl) -2,3-dimethylbutane, or M ₄ N is That is, a solution containing NDGA and potassium hydroxide in methanol in a reaction flask is prepared. Next, dimethyl sulfate is added to the reaction flask and the reaction is allowed to proceed. The reaction finally precipitates the product by quenching with water. The precipitate is isolated by filtration and dried in a vacuum oven. The compound is then dissolved in a solution of methylene chloride and toluene and then purified through an alumina column. The solvent is removed on a rotary evaporator and the solid is resuspended in isopropanol and purified by filtration. The filter cake is dried in a vacuum oven. The resulting tetra-O-methyl NDGA (M ₄ N) is crystallized by refluxing the filter cake in isopropanol and reisolating the crystals by filtration.

In some embodiments of the invention, certain NDGA derivatives of the invention, such as G ₄ N, also known as meso-1,4-bis [3,4- (dimethylaminoacetoxy) phenyl]-(2R, 3S) -Similar compounds having dimethylbutane or tetra-dimethylglycinyl NDGA, or its hydrochloride and amino acid substituents can also be prepared according to conventional methods such as described in US Pat. No. 6,417,234.

  Manufacture of therapeutic compositions

  The present invention provides compositions comprising catecholbutane as an active pharmaceutical ingredient (“API”), such as NDGA compounds and NDGA derivatives, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. To do. Typically, the compositions of the present invention contain less than about 0.1% up to about 99% API, ie catecholbutane, such as the NDGA compounds and NDGA derivatives described herein, from about 0.1% to about 99%. Optionally, the present invention contains from about 2% to about 90% API.

The present invention further provides catecholbutane, such as NDGA compounds, such as NDGA derivatives, such as M ₄ N, from about 1 mg / mL to about 200 mg / mL, or from about 10 mg / mL to about 175 mg / mL, or from about 20 mg / mL to about Compositions are provided such that they are present at a concentration of 150 mg / mL, or about 30 mg / mL to about 125 mg / mL, or about 40 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 75 mg / mL. In one embodiment, the NDGA compound is about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL in the compositions described herein. mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 75 mg / mL It is present at a concentration of mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL or about 200 mg / mL.

The present invention further provides catecholbutane, eg, NDGA compounds, eg, NDGA derivatives, eg, M ₄ N, of about 1 mg / g to about 250 mg / g, or optionally about 20 mg / g to about 200 mg / g, or about As present at concentrations ranging from 40 mg / g to about 180 mg / g, or from about 60 mg / g to about 160 mg / g, or from about 80 mg / g to about 130 mg / g, or from about 50 mg / g to about 100 mg / g. A composition is provided. In one embodiment, the compounds of the invention are present in the compositions described herein at concentrations of about 133 mg / g, about 185 mg / g, about 200 mg / g and about 250 mg / g. Exemplary amounts of API in the composition include, but are not limited to, about 20 mg / g, about 50 mg / g, about 75 mg / g, about 100 mg / g, about 120 mg / g, about 130 mg / g, about 140 mg / g. g, about 150 mg / g, about 175 mg / g or 200 mg / g.

  Expressed as an alternative, another embodiment of the composition of the present invention comprises less than about 0.1 mg up to about 200 mg of API, eg, about 10 mg, about 20 mg, about 25 mg, About 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg or about 200 mg of API may be included.

  In one embodiment of the invention, a composition comprising an API and a carrier is provided, wherein the API is catecholbutane and the carrier is a solubilizer and / or enhancer for dissolving the API. Containing a dosage form, wherein the solubilizers and / or excipients are: (a) a water-soluble organic solvent, such as a PG (propylene glycol) or PEG (polyethylene glycol) compound, provided that the PEG compound Are for example PEG 300, PEG 400 or PEG 400 monolauric acid; (b) cyclodextrins, such as modified cyclodextrins; (c) ionic, non-ionic or amphiphilic surfactants, such as Tween 20, Tween 80 or TPGS, glycerol monooleic acid and esterified fatty acids; (d) modified cellulose E.g. EC, HPMC, MC or CMC; and (e) water-insoluble liquids such as oils, waxes or fat emulsions such as Intralipid, but if the composition contains castor oil, it contains beeswax or carnauba wax Contains at least one of the above substances (a) to (e), but may contain more.

  In one embodiment, the present invention provides a composition as described above, which is a liquid composition suitable for oral administration. In another embodiment, the present invention provides a composition as described above, which is a solid composition suitable for oral administration.

  In yet another embodiment, the present invention does not contain (a) one of glycerin or glycine; or (b) white petrolatum; or (c) xanthan gum if the composition contains propylene glycol. , Providing a composition as described above.

  In yet another embodiment, the present invention provides a composition as described above that does not contain xanthan gum when the composition contains a nonionic surfactant.

  In yet another embodiment, the present invention provides a composition as described above that does not contain PEG 8000 or BHT when the composition contains PEG 400.

  Among the preferred water-soluble organic solvents are ethanol, benzyl alcohol, dimethylacetamide, PVP, PG and PEG compounds such as PEG 300, PEG 400 or PEG 400 monolauric acid. The PEG compound in the composition of the present invention is about 5% to about 100%, or about 5% to about 60%, or about 10% to about 90%, or about 20% to about 80%, or about 30%. Present in an amount of about 70%, or about 40% to about 60%, and all concentrations are given in volume / volume (v / v) ratios. PG may be present at a concentration of about 2.5% to about 100% (v / v).

  The concentration of PEG compound in the composition of the invention will vary depending on other solubilizers or diluents or excipients that are also present. For example, PEG 300, PEG 400 or PEG 400 monolauric acid of the present invention is about 5%, about 10%, about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35%. About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% All these concentrations are given as a volume / volume (v / v) ratio.

  The present invention also provides compositions of catecholbutane or NDGA compounds in cyclodextrins, including modified cyclodextrins. The cyclodextrins described herein may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and modified cyclodextrins include, for example, HP-β-CD and SBE-β-CD. May be included. In one embodiment, the composition of the present invention comprises from about 5% to about 80%, or from about 10% to about 70%, or from about 20% to about 60%, or from about 30% to about 50% modified cyclodextrin. Concentrations in%, all these concentrations are given in weight / volume (w / v) ratios.

  In yet another embodiment, the modified cyclodextrin, such as HP-β-CD, is about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35% in the composition. , About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% or about 75%, all of which are weight / volume (w / It is indicated by the ratio of v).

  Other pharmaceutically acceptable carriers or excipients that may be used alone or in combination with others include ionic, nonionic or amphiphilic surfactants such as Cremophor EL. Polysorbates that are nonionic surfactants, such as polysorbate 20 and polysorbate 80 commercially available as Tween 20 or Tween 80, TSGS that are amphiphilic surfactants, and the like. Another example of a suitable surfactant includes, but is not limited to, glycerol monooleic acid and esterified fatty acids, such as those typically produced by transesterification of vegetable oils, from Gattefosse Corp., Paramus, NJ, USA Labrafil (registered trademark: hereinafter omitted, “Labrafil” is a registered trademark), Labrasol (registered trademark: hereinafter, “Labrasol” is a registered trademark), and Gelucire (registered trademark: hereinafter, omitted) However, “Gelucire” is a registered trademark.) And is available in several categories and grades. The surfactant is in any desired effective amount, for example, from about 1% (v / v) to about 100% (v / v), preferably from about 9% (v / v) to about 80% (v / v). v), more preferably present as a concentration of about 10% (v / v) to about 50% (v / v). As a specific example, preferred concentrations of nonionic surfactants are Tween 20 at a concentration of about 9% (v / v) to about 100% (v / v), and about 33% (v / v) to about 100% (v / v) Tween 80. The total ratio of surfactant is the volume ratio (v / v).

  Another pharmaceutically acceptable carrier or excipient that may be used alone or in combination with others is a modified cellulose, such as EC, HPMC, MC and CMC. The modified cellulose is present in any desired effective amount, eg, at a concentration of about 0.1% to about 25%, or about 0.5% to about 7.5%, or about 1.0% to about 5%. it can. In particular examples, EC may be present at a concentration of about 5% to about 20%; HPMC may be present at a concentration of about 0.5% to about 1%; MC is about 1% CMC may be present at a concentration of about 1% to about 4%. The ratio of modified cellulose is the weight per volume (w / v).

  In another embodiment, the present invention provides a composition as described above comprising a PEG compound, such as PEG 400, and a surfactant, such as Tween 20, as a solubilizer and / or excipient.

  Another pharmaceutically acceptable carrier or excipient that may be used alone or in combination with others is a water-insoluble lipid, such as an oil, fat emulsion or wax. The water-insoluble liquid carrier can be present in any desired effective amount, eg, at a concentration of about 10% to about 100%, or about 15% to about 85%, or about 25% to about 75%.

  Non-limiting examples of oils include corn oil, olive oil, peppermint oil, soybean oil, sesame seed oil, mineral oil and glycerol. In one embodiment, the oil is present at a concentration of about 10% (v / v) to about 100% (v / v). As the mixed fat emulsion composition, for example, the above-mentioned Intralipid emulsion can be used. In various embodiments, the mixed fat emulsion may be present at a concentration of about 10% (w / v) to about 30% (w / v); and preferably about 20% (w / v). Non-limiting examples of suitable waxes are beeswax and carnauba wax. In one embodiment, the wax is present at a concentration of about 5% (w / w) to about 50% (w / w).

  Water insoluble carriers may be used in combination with one or more of water soluble carriers such as PEG compounds and surfactants such as either Tween 20 or Tween 80. As another example, the compositions of the present invention comprise solubilizers and / or excipients such as oils and surfactants such as peppermint oil and Tween 20 or combinations of oil and PEG compounds such as peppermint oil and PEG 400, Containing.

  In another embodiment, the composition of the present invention contains an oil, a surfactant and a PEG compound, such as peppermint oil, Tween 20 and a PEG 400 compound.

  As another example, the compositions of the present invention include oils or oils and waxes such as peppermint oil and sesame oil, soybean oil and beeswax or olive oil and beeswax combinations. Beeswax may be present at concentrations ranging from about 5% to 50% (w / w).

  Alternatively, in another embodiment, Tween 80 may be replaced with Tween 20, and PEG 300 or PEG 400 monolauric acid may be replaced with PEG 400, and either of the oils may be peppermint oil, soybean oil And olive oil.

  Still other excipients include glycerol monooleic acid and various types of esterified fatty acids such as Labrafil, Labrasol and Gelucire products. These are typically classified as surfactants or emulsifiers, but Labrasol and Gelucire products are also used as bioavailability enhancers.

Labrafil, particularly Labrafil M 1944 CS, can be used as an excipient with an API, such as M ₄ N, where the API is dissolved at a concentration of about 5 mg / mL to about 500 mg / mL. The final product may be a solution, suspension or solid.

Labrasol can be used as an excipient with an API, such as M ₄ N, which dissolves at a concentration of about 1 mg / mL to about 500 mg / mL. The final product may be a solution, suspension or solid.

Gelucire, especially Gelucire 44/14, can be used as an excipient with an API, such as M ₄ N, which dissolves at a concentration of about 30 mg / mL to about 500 mg / mL. The final product is a solid.

Glyceryl monooleic acid can be used as an excipient with an API, such as M ₄ N, which dissolves at a concentration of about 0.1 mg / mL to about 500 mg / mL. The final product may be a solution, suspension or solid.

Various carrier component combinations may be used with the API as described above. One non-limiting example of such an embodiment is 25% (w / v) PEG 300, 30% (w / v) HP-β-CD, “water for injection” to the animal as a carrier balance. A composition of 10 mg / mL M ₄ N in (“WFI”, ie, which indicates the grade of water found in the pharmaceutical industry). In this preferred embodiment, HP-β-CD has a degree of substitution of about 6-8, although other substitutions in other embodiments are also encompassed within the scope of the invention as described above.

  Other excipients and additives, such as bioavailability enhancers, may be used in combination with any of the carriers in the compositions of the present invention. Suitable bioavailability enhancers include, but are not limited to, eugenol, cinnamaldehyde, lecithin, naringenin, naringin, and piperine (also called piperine), for example.

  Solubilizers described herein as long as the catecholbutane described herein dissolves and remains in solution, suspension, or maintained in a composition in semi-solid or solid form. Alternatively, one or more of a diluent or excipient may be added to the composition to optimize its delivery to a subject in need of such treatment.

  Other pharmaceutically acceptable carriers or excipients suitable for use in the present invention are described in various publications. Examples of useful carriers or excipients include, for example, Gennaro, AR (2003); Ansel, HC et al. (2004); Rowe RC) et al. (2003); and Garg, S. et al. (2001).

  Liquid form compositions may include a buffer, which is selected according to the desired use of the catecholbutane or NDGA compound, eg, NDGA derivative, and includes other materials suitable for the intended use. Good. Appropriate buffers are readily selectable by one skilled in the art, a wide variety of which are known in the art and suitable for the intended use.

  Method of treatment

  A composition containing catecholbutane, such as an NDGA compound, such as an NDGA derivative, as a therapeutic agent in a subject in need of treatment in any number of diseases for which such catecholbutane or NDGA compound or derivative can be used, or Can be used for such treatment.

  The present invention provides methods and compositions for the treatment of diseases such as proliferative diseases such as benign and malignant cancers, psoriasis and premalignant conditions and neoplasia such as intraepithelial neoplasia or dysplasia. . The present invention also provides for the treatment of diabetes, eg, type I and type II diabetes, obesity and complications arising from such, eg, cardiovascular disease, stroke and hypertension. The present invention further provides treatment of inflammatory diseases such as rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, chronic obstructive pulmonary disease (COPD) and other immune system related diseases. . Furthermore, the present invention also provides for the treatment of neurological diseases such as diseases of the central nervous system and neurodegenerative diseases such as Alzheimer's disease, dementia, amyotrophic lateral sclerosis (ALS) and Parkinson's disease. To do. In yet another embodiment, the present invention provides for the treatment of infectious diseases, eg, viral infections including viruses that require Sp1 binding for transcription or replication. Examples of such viruses that require Sp1 binding include HIV, HTLV, HPV, HSV, HBV, EBV, varicella-zoster virus, adenovirus, parvovirus and JC virus.

  A variety of animal hosts, including human and non-human animals, can be treated according to the subject method. In general, such hosts are “mammals” or “mammalian” and these terms are organisms classified as mammals, such as carnivores (eg dogs and cats), rodents (eg guinea pigs and rats). ), And other mammals such as cows, goats, horses, sheep, rabbits, pigs and primates (eg, humans, chimpanzees and monkeys). In many embodiments, the host is a human. Animal models are advantageous for experimental investigations, such as providing models for the treatment of human diseases. Furthermore, the present invention is also applicable to veterinary medicine.

  Formulation, dose and route of administration

  In one embodiment of the invention, an effective amount of a composition of the invention is administered to a host, where “effective amount” means a dose sufficient to achieve the desired result. In some embodiments, for example, the desired result is at least suppression of progression of neoplasia or dysplasia.

The present invention further provides active pharmaceutical ingredients, such as catecholbutane, such as NDGA compounds, such as NDGA derivatives, such as M ₄ N, for example from about 1 mg / kg to about 600 mg / kg (about less than the body weight of an animal such as a human). Compositions are provided for administration to animals at an oral dose of from 1 mg / kg to about 600 mg / kg). Optionally, the animal is 1 mg / kg, or 50 mg / kg, or 100 mg / kg, or 150 mg / kg, or 200 mg / kg, or 250 mg / kg, or 300 mg / kg, or 350 mg / kg, or 400 mg / kg, Alternatively, it may be treated with 450 mg / kg, or 500 mg / kg, or 550 mg / kg. Such doses may be administered once or repeatedly over several days, weeks or months. Alternatively, such doses may be spread out over a period of time depending on the subject's health, the subject's susceptibility, the extent of the disease to be treated, the age of the subject, and the like.

  In one embodiment, the therapeutic compositions described herein are prepared by first dissolving the active pharmaceutical ingredient in a solubilizer, optionally with stirring and heating. Other excipients are added to the solubilized mixture to achieve the desired ratio with respect to substrate and stability conditions. In another embodiment, the active pharmaceutical ingredient is not actually dissolved in the solubilizer and may simply be uniformly dispersed in the suspension. In another embodiment, the solubilized composition may be lyophilized and used in powder form. The final oral composition may be in the form of a liquid solution or suspension, or may be in the form of a solid powder, tablet or capsule.

  As stated above, the appropriate dose to be administered depends on the subject to be treated, eg, the general health of the subject, the age of the subject, the condition or condition of the disease, the weight of the subject, the extent of the disease, eg, the size of the tumor, etc. It is a thing. Generally, about 0.1 mg to about 500 mg or less (500 mg or less) may be administered to a child and about 0.1 mg to about 5 grams or less may be administered to an adult. The active agent can be administered in a single or more typically in multiple doses. The preferred dosage for a given drug can be readily determined by those skilled in the art by various means. Other effective doses are readily determined by those skilled in the art by routine trials preparing dose response curves. The amount of drug will of course vary depending on the particular drug used.

  The frequency of administration of the active agent is determined by the nurse based on age, weight, disease status, health status and patient responsiveness, as with doses. That is, the drug may be administered once or more, as appropriate, as determined daily, weekly, monthly or routinely. The drug is administered intermittently, for example over a period of days, weeks or months, after which it is not re-administered until after a certain period of time, eg 3 or 6 months, and then again for days or weeks Or it may be administered over a period of several months.

  In pharmaceutical dosage forms, the active agents may be administered alone or in appropriate association and combination with other pharmaceutically active agents or therapeutic agents such as other small molecules, antibodies or protein therapeutics.

Furthermore, if desired, the carrier or excipient may contain minor amounts of auxiliary substances such as pH adjusting and buffering agents, osmotic pressure adjusting agents, stabilizers, wetting agents or emulsifiers. The actual methods of preparing such dosage forms are known or known to those skilled in the art. For example, ^{Remington's Pharmaceutical Sciences, 20 th ed} ., Can see Mack Publishing Co. Rawlins, EA, the (1997). The composition or formulation to be administered will in any case contain a certain amount of API suitable to achieve the desired condition in the subject to be treated.

  Kits with multiple or unit doses of active agents are encompassed by the present invention. In such a kit, in addition to a container holding multiple or unit doses of a composition containing catecholbutane, eg, an NDGA compound, eg, an NDGA derivative, the use of the agent in treating a pathological condition of interest and An informational package insert with instructions describing the benefits associated therewith is included. Optionally, an applicator for administration of the composition of the present invention is included in each kit.

  The examples described below are exemplary in nature and should not be construed as limiting the invention.

Example 1. Formulation containing M ₄ N in HP-β-CD and / or PEG 300.

In this example, M ₄ N was prepared as described in PCT / US2004 / 016117 and solubilized in a solubilizer. The resulting solution was optionally mixed with excipients and / or diluents. Solubilizers and excipients may be used interchangeably or in combination with each other. One solubilizer or excipient used was endotoxin controlled hydroxypropyl-β-cyclodextrin (“HP-β-CD”) (Catalog Number RDI-82004HPB, Lot Number H3N188P available from Research Diagnostics, Inc. (Flanders, NJ, USA). Another solubilizer or excipient used was PEG 300 (catalog number P0108, lot number TB1228) (Gardena, CA, USA) obtained from Spectrum Chemicals, Inc.

In one embodiment of the invention, HP-β-CD and PEG 300 are present in a single formulation. To make this formulation, M ₄ N was first dissolved in PEG 300 to form a M ₄ N solution in PEG 300 (“M ₄ N / PEG 300”). The M ₄ N / PEG 300 solution is then added to the previously prepared HP-β-CD solution to add the M ₄ N solution in PEG 300 and HP-β-CD (hereinafter referred to as the “CPE” formulation). Formed.

  In preparing the HP-β-CD solution, volume expansion must be taken into account. For example, for a 40% (w / v) HP-β-CD solution, a volume expansion of 0.7 mL / g (ie, 0.7 mL of water is replaced per gram of added HP-β-CD) must be considered. .

  In preparing a 100 mL solution of 40% HP-β-CD for use as a solubilizer and / or excipient, 65 mL of WFI was placed in a glass beaker with a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 40 grams of HP-β-CD is slowly added to the stirring WFI, with HP-β-CD crystals being placed in the beaker using a spatula so that the HP-β-CD faces the center of the beaker. Prevents sticking to the wall. The HP-β-CD solution was stirred for about 24 hours or until it was visually determined that the HP-β-CD was completely dissolved. The resulting solution was about 93 mL. About 7 mL of WFI was added to this solution to 100 mL to prepare a final solution of about 40% HP-β-CD. The final solution was stirred for about 1 hour and stored at room temperature in the dark. The desired volume or concentration may be obtained by scaling up or down this method of preparing a modified cyclodextrin solution. Other concentrations or other modified cyclodextrin solutions may be similarly prepared, for example, by replacing HP-β-CD with other modified cyclodextrins adjusted to the appropriate concentration in the method described above. .

A 10 ml solution of M ₄ N at a concentration of about 10 mg / mL in 40% HP-β-CD was prepared as follows. About 10 ml of 40% HP-β-CD solution was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 10 mg of M ₄ N was slowly added to 40% HP-β-CD in the center of the beaker using a spatula. The M ₄ N / HP-β-CD mixture was stirred for 2 hours or until all the M ₄ N was suspended uniformly and no lumps were present. The M ₄ N / 40% HP-β-CD mixture is optionally about 80 minutes at 80 ° C. (or longer if a larger volume of solution is desired, eg M ₄ N / 40% HP-β -M ₄ N was completely dissolved by heating for 1 hour at 80 ° C. for 100 ml of the CD mixture) or longer if necessary. M ₄ N / 40% HP for the presence of undissolved M ₄ N by holding the beaker against a white background and then a dark background and then probing for the presence of particulate matter. -A β-CD mixture or solution was observed. The final M ₄ N / HP-β-CD solution was stored at room temperature in the dark. The desired volume or concentration of M ₄ N may be obtained by scaling up or down the method. Formulations containing M ₄ N in other cyclodextrin solutions may be similarly prepared, for example, by replacing HP-β-CD in the above method with other cyclodextrins. The results shown in Table 1 reveal that M ₄ N remained in solution after cooling at concentrations of 1 mg / mL and 10 mg / mL in 40% HP-β-CD formulations for longer than 7 days. .

A 100 ml solution of M ₄ N at a concentration of about 25 mg / mL in PEG 300 was prepared as follows. About 100 ml of PEG 300 was added to a glass beaker containing a stir bar. A glass beaker was placed on the magnetic plate and the stir bar was set to stir at medium speed. About 2.5 g of M ₄ N was slowly added to PEG 300 in the center of the beaker using a spatula to prevent M ₄ N from sticking to the beaker wall. The M ₄ N / PEG 300 mixture was stirred for 24 hours or until all M ₄ N was dissolved or homogeneously suspended and no lumps were present. The M ₄ N / PEG 300 mixture is optionally about 60 minutes at 60 ° C. (or longer if a larger volume of the solution is desired, eg 1 ml at 60 ° C. for 500 ml of M ₄ N / PEG 300 mixture. Time), or by heating for a longer time if necessary, M ₄ N was completely dissolved. Whether the undissolved M ₄ N is present by holding the beaker against a white background and then a dark background and then probing for the presence of particulate matter, the M ₄ N / PEG 300 mixture or The solution was observed. After all M ₄ N is observed to dissolve, the resulting M ₄ N / PEG 300 solution is formed immediately or before the 48 hour expiration date, or crystals or other precipitates are formed. Used before. If crystals formed, the M ₄ N / PEG 300 solution was again heated with stirring on a high temperature magnetic plate at 60 ° C. for 1 hour until all the M ₄ N was redissolved in the solution. The final M ₄ N / PEG 300 solution was stored at room temperature in the dark. The desired volume or concentration of M ₄ N may be obtained by scaling up or down the method. Formulations containing M ₄ N in other PEGs may be similarly prepared, for example, by replacing PEG 300 in the method described above with PEG 400 or PEG 400 monolauric acid.

100 mL stock solution of a formulation containing 50% PEG 300 (v / v), 20% HP-β-CD (w / v) and 12.5 mg M ₄ N was stirred on a magnetic plate. 50 ml of 40% HP-β-CD solution (prepared as described above) prepared in advance was added to a glass beaker containing a rod, and the stirring rod was stirred at medium speed to prepare 50 mL of M prepared in advance. _{A 4} N / PEG 300 solution (prepared as described above) was prepared by slowly adding, for example, at a rate of about 10 mL / min. M ₄ N / PEG 300 was added using a pipette in the middle of the beaker to prevent it from sticking to the beaker wall and to ensure complete dissolution. The addition of M ₄ N / PEG 300 to the HP-β-CD solution was initially in the form of a white solution, but finally became clear by continuous stirring. This procedure may be scaled up or down as appropriate to achieve the desired volume and concentration of M ₄ N / PEG 300 and HP-β-CD. The stock solution was filter sterilized using a 0.22 μm PVDF membrane, such as a pre-sterilized vacuum driven disposable bottle top filter membrane (Catalog No. SCGV T05 RE) (Billerica, MA, USA) available from Millipore. The filtration procedure was driven by vacuum and the filtrate was collected in a pre-sterilized 250 mL volume glass bottle. The bottle was then sealed and stored at room temperature in the dark. A stock solution of M ₄ N / PEG 400 or M ₄ N / PEG 400 monolauric acid in HP-β-CD can be similarly prepared by replacing PEG 300 with PEG 400 or PEG 400 monolauric acid in the manner described above. .

A stock solution of M ₄ N / PEG 300 / HP-β-CD, M ₄ N / PEG 400 / HP-β-CD or M ₄ N / PEG 400 monolauric acid / HP-β-CD prepared in the above-described embodiment is It can be diluted prior to use in vitro or for administration to animals. If dilution is required, the stock solution is preferably diluted with WFI instead of saline, eg, to keep osmotic pressure low. Approximately 50 mL of stock solution was added to a glass vial to prepare a 100 mL 1: 1 dilution of stock solution in WFI. Approximately 50 mL of WFI was added to 50 mL of stock solution in the vial to form a diluted solution. The glass vial was closed and the diluted solution was mixed well by inverting the vial several times. The diluted solution was filter sterilized using a 0.22 μm PVDF membrane, such as a pre-sterilized vacuum driven disposable bottle top filter membrane (Catalog Number SCGV T05 RE) (Billerica, MA, USA) available from Millipore. . The filtration procedure was driven by vacuum forcing and the filtrate was collected in pre-sterilized 250 mL capacity glass bottles. The bottle was then sealed and stored at room temperature in the dark. This method may be scaled up or down to achieve the required volume or dilution, eg, 1: 2 or 1: 4 dilution.

  A formulation suitable for use as a placebo control containing 50% PEG 300 and 20% HP-β-CD can be prepared as follows. To prepare a 100 mL solution of placebo or control formulation, add approximately 50 mL of 40% HP-β-CD to a glass beaker with a stir bar on a magnetic plate. The magnetic plate is set to stir the HP-β-CD solution at medium speed. About 50 mL of PEG 300 is slowly added to 50 mL of HP-β-CD in a glass beaker using a pipette at the center of the beaker so that PEG 300 does not stick to the beaker wall. Stir the mixture for about 1 hour or until thoroughly mixed. The placebo formulation is filter sterilized using a 0.22 μm PVDF membrane filter driven by vacuum force. Collect the filtrate in pre-sterilized 250 mL capacity glass bottles. The glass bottle was sealed and stored at room temperature in the dark. This formulation may be scaled up or down as needed to achieve the desired concentration and volume. Furthermore, PEG 300 may be replaced with PEG 400 or PEG 400 monolauric acid if desired.

HP-β-CD and propylene glycol (“PG”), hydroxylpropyl methylcellulose (HPMC), prepared in a formulation containing HP-β-CD and / or PEG 300, PEG 400, and according to the above or similar methods, The results of solubility of M ₄ N in formulations containing carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP) or Tween 80 and the properties of the resulting formulations are shown in Tables 1-5, where N is “No” ", Y indicates" Yes ".

All formulations were tolerated by centrifugation for 24 hours at 4 ° C. and 5 minutes at 5000 rpm, and no visible precipitate formed. A formulation containing a stock solution of 50% PEG 300, 20% HP-β-CD, 12.5 mg / mL M ₄ N was tolerated at 4 ° C. for at least 4 months. The same stock solution dilutions prepared at 1: 1 or 1: 2 dilutions were also tolerated at 4 ° C. for at least 4 months.

Similarly, M ₄ N may be solubilized in other solubilizing agents such as water-soluble organic solvents including ethanol, PVP (polyvinyl pyrrolidone), propylene glycol or glycerol.

Example 2 Effect of M ₄ N in DMSO or in complex PEG 300 / HP-β-CD formulations on tumor cell growth and cell death in culture

M ₄ N (hereinafter referred to as “CPE formulation”) in 10% (w / v) HP-β-CD and 25% (v / v) PEG 300, 30% (w / v) HP-β-CD and M ₄ N in 25% (v / v) PEG 300 (hereinafter referred to as “CPE25 / 30 formulation”), and 27% (w / v) HP-β-CD and 33% (v / v) PEG Two tumor cell lines of M ₄ N in 300 (hereinafter referred to as “CPE33 / 27 formulation”): HPV-18 positive human cervical cancer cell line HeLa and HPV negative human cervical cancer cell line C-33A Were tested for effects on cell death and proliferation. Both tumor cell lines were treated with increasing amounts of M ₄ N: 0 μM, 20 μM, 40 μM, 60 μM and 80 μM with DMSO or CPE formulations for 72 hours. Each formulation is in a total volume of 1% growth medium (minimum essential medium containing L-glutamine supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1 × non-essential amino acid solution and 1000 IU / mL penicillin / 1000 μg / mL streptomycin solution). Added. Control cells were grown under the same conditions and left untreated. 72 hours after treatment or untreatment, the total number of cells and the number of viable cells in each sample were counted by trypan blue dye exclusion. The cell growth rate and dead cell ratio of each sample was analyzed. The results of this experiment are shown in FIGS. 1 and 2 and Tables 6-12.

FIG. 1 graphically illustrates the ratio of treated / untreated cells plotted against increasing concentrations of M ₄ N in either DMSO or CPE formulations for treatment of C-33A cells and HeLa cells. Is. FIG. 2 shows the ratio of dead cells plotted against increasing concentrations of M ₄ N in either DMSO or CPE formulations for the treatment of two cancer cell lines, namely C-33A and HeLa cells in culture. Is shown in a graph.

The results show that DMSO alone in the absence of M ₄ N has a significant antiproliferative effect and some degree when measured in% of dead cells, as compared to untreated controls, for both tested tumor cell lines. It has a toxic effect. In contrast, the CPE formulation alone has an antiproliferative and very little toxic effect on the two tumor cell lines when compared to the untreated control.

Cell growth rates were both after treatment of M ₄ N in either DMSO or CPE formulations when compared to non-M ₄ N treated controls in the same formulation (ie 0 μg / mL or 0 μg of M ₄ N). Decreased in all cell lines. Indeed, the antiproliferative effect appeared to be dose dependent on M ₄ N in the CPE formulation. In CPE formulations, for example, about 20 μM or 7.2 μg / mL of M ₄ N was found to be sufficient to provide about 50% inhibition of cell proliferation for both tumor cell types. Further increasing the concentration of M ₄ N in the CPE formulation further increased the antiproliferative effect on both cell types.

Generally, higher doses of _{M 4} N from in either DMSO formulation or CPE formulation induced higher percentage of cell death for both C-33A cells and HeLa cells. However, _{M 4} N in DMSO formulations was highly toxic than to cells than the corresponding concentration of _{M 4} N in CPE preparation. The highest concentration of M ₄ N tested in the DMSO formulation (80 μM or 28.7 μg / mL) promoted cell death in about 40% of the cell population, but the same concentration of M ₄ N in the CPE formulation In experiments, only about 20% of the cell population promoted cell death.

These results were found to be reproducible in both cell lines. Data from this study show that M ₄ N in CPE formulations has the same ability to stop cell growth as M ₄ N in DMSO formulations, but induces lower cytotoxicity than DMSO formulations. ing.

  Data were collected from successive time points to test the effectiveness of the CPE formulation over time. Data showed that after a 12 month period of storage at 2-8 ° C., the CEP formulation was as effective as at the time of new manufacture. Cell viability remained the same for 12 months when the CPE formulation was stored. Cell death and proliferation rates remained within the same range.

  Data was collected to compare the original CPE formulation with the new CPE 25/30 formulation. Tests were performed at 0 and 3 months to test the efficacy of the formulation over time and to test how well the new formulation functions compared to the old. The data show that the CPE 25/30 formulation is as effective in inhibiting tumor cell growth as the original CPE formulation. Cell viability was similar among HeLa cells treated with various concentrations of drug using either CPE or CPE25 / 30 formulations. Cell death and proliferation remained within the same range even when viewed over time.

  Information was collected comparing the original CPE formulation to the CPE33 / 27 formulation at time zero for HeLa cells. The data showed that CPE33 / 27 had some influence on HeLa cells in cell viability, ratio of dead cells and proliferation rate.

Example 3 Multiple lots of M ₄ N can be used to achieve the same result.

Different lots of M ₄ N were tested to show the effectiveness of different lots of drugs. HeLa cells were treated with increasing amounts of M ₄ N: 0 μM, 20 μM, 40 μM, 60 μM and 80 μM with the CPE formulation for 72 hours. Each formulation is in a total volume of 1% growth medium (minimum essential medium containing L-glutamine supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1 × non-essential amino acid solution and 1000 IU / mL penicillin / 1000 μg / mL streptomycin solution). Added. Control cells were grown under the same conditions and left untreated. 72 hours after treatment or untreatment, the total number of cells and the number of viable cells in each sample were counted by trypan blue dye exclusion. The cell growth rate and dead cell ratio in each sample was analyzed. The results of this experiment are shown in Tables 13 and 14. These results show that the efficacy of the drug remains the same regardless of the M ₄ N lot used.

Example 4 Solubility of M ₄ N in modified cellulose

  Preparation of a 10 mL solution of 50% HP-β-CD (w / v) and 0.5% hydroxylpropylmethylcellulose (“HPMC”) (w / v) for use as a solubilizer and / or excipient At that time, 5.9 mL of WFI was put into a glass beaker containing a stirring bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. 5 grams of HP-β-CD was slowly added to the stirring WFI, using a spatula to direct HP-β-CD to the center of the beaker. The HP-β-CD solution was stirred for about 24 hours or until it was visually determined that the HP-β-CD was completely dissolved. The resulting solution was about 9.4 mL. About 0.6 mL of WFI was added to this solution to make 10 mL, and a 50% HP-β-CD solution (w / v) was prepared. 50 milligrams of HPMC was added to the 50% HP-β-CD solution and stirred for about 1 hour or until it was visually determined that the HPMC had dissolved. The final solution was stirred for about 1 hour and then stored at room temperature in the dark. The desired volume or concentration of the HP-β-CD / HPMC solution may be obtained by scaling up or down this method of preparing modified cyclodextrins containing modified cellulose. Other modified cyclodextrin / modified cellulose solutions may be similarly prepared, for example, by replacing HP-β-CD with other modified cyclodextrins or HPMC with other modified celluloses in the manner described above.

A 10 mL M ₄ N solution at a concentration of about 10 mg / mL in 50% HP-β-CD / 0.5% HPMC was prepared as follows. About 10 mL of 50% HP-β-CD / 0.5% HPMC solution was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 100 mg of M ₄ N was slowly added to 50% HP-β-CD / 0.5% HPMC in the center of the beaker using a spatula. The M ₄ N / 50% HP-β-CD / 0.5% HPMC mixture was stirred for 24 hours or until all M ₄ N was uniformly suspended and no lumps were present. The M ₄ N / 50% HP-β-CD / 0.5% HPMC mixture is about 90 ° C. for about 30 minutes (or longer if a larger volume of solution is desired, eg 500 mL of M ₄ N / The 50% HP-β-CD / 0.5% HPMC mixture was heated at 90 ° C. for 1 hour) or longer if necessary to ensure complete dissolution of M ₄ N. Whether or not undissolved M ₄ N is present by holding the beaker against a white background and then a dark background and then probing for the presence of particulate matter, M ₄ N / 50% HP− A β-CD / 0.5% HPMC mixture was observed. The final M ₄ N 50% HP-β-CD / 0.5% HPMC solution was stored at room temperature in the dark. M ₄ N is dissolved in this 50% HP-β-CD / 0.5% HPMC formulation at concentrations of 1 mg / mL and 10 mg / mL when heated at 90 ° C. and remains as a solution after cooling. The stability at room temperature was longer than 7 days. M ₄ N did not dissolve even at 90 ° C. at a 50 mg / mL concentration in this same formulation.

The desired volume or concentration of M ₄ N may be obtained by scaling up or down the method described above. Formulations containing M ₄ N in other cyclodextrin / cellulose solutions are similar, for example, by replacing HP-β-CD with other cyclodextrins in the method described above, or replacing HPMC with other modified celluloses. May be prepared.

  In preparing a 10 mL solution of 5% ethylcellulose in ethanol (w / v) (“EC”) for use as a solubilizer and / or excipient, 10 mL of 100% in a glass beaker with a stir bar. Ethyl alcohol (“EtOH”) was charged and covered with a circular Tefron (registered trademark: hereinafter, “Tefron” is a registered trademark) cover. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. 500 milligrams of EC was slowly added to the stirring ethanol, using a spatula to direct the EC to the center of the beaker to prevent the EC powder from sticking to the beaker wall. The EC solution was stirred for about 2 hours or until it was visually determined that the EC was completely dissolved. The final solution was stored at room temperature in the dark.

  The desired volume or concentration may be obtained by scaling up or down this method of preparing a modified cellulose solution. Other modified cellulose solutions may be similarly prepared, for example, by replacing EC with other modified cellulose in the method described above.

A 10 mL solution of M ₄ N (“EC formulation”) at a concentration of about 20 mg / mL (w / v) in 5% EC (w / v) was prepared as follows. About 10 mL of 5% EC solution prepared as described above was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed and covered with a circular Teflon cover. About 200 mg of M ₄ N was slowly added to the 5% EC formulation in the center of the beaker using a spatula to prevent M ₄ N from sticking to the beaker wall. The M ₄ N / EC mixture was stirred for 2 hours or until all M ₄ N was dissolved or homogeneously suspended and no lumps were present. M ₄ N / EC mixture at about 60 ° C. for about 30 minutes (or longer if a larger volume of solution is desired, eg 1 hour at 60 ° C. for a 500 mL M ₄ N / EC mixture) or required In response to this, M ₄ N was completely dissolved by heating for a longer time. The M ₄ N / EC mixture or whether the undissolved M ₄ N is present is determined by holding the beaker against a white background and then a dark background and then probing for the presence of particulate matter. The solution was observed. The final M ₄ N / EC solution was stored at room temperature in the dark.

The desired volume or concentration of M ₄ N may be obtained by scaling up or down the above method, and the heating temperature may be increased or decreased to achieve dissolution of M ₄ N. Formulations containing M ₄ N in solutions of other modified celluloses such as HPMC, MC and CMC may be similarly prepared. The results of solubility of M ₄ N in modified cellulose are shown in Table 15.

The results show that M ₄ N was not soluble in 2.3% (w / v) HPMC at any concentration tested or by heating to 90 ° C. M ₄ N formed a suspension in 1% (w / v) HPMC at a concentration of 10 mg / mL. This suspension was stable at room temperature for less than 2 days.

In the presence of HP-β-CD (50% w / v) and HPMC (0.5% w / v), M ₄ N is solubilized at 90 ° C. and at concentrations of 1 mg / mL and 10 mg / mL, It remained in solution during cooling. These solutions were stable at room temperature for more than 7 days. M ₄ N dissolves in the same HP-β-CD (50% w / v) and HPMC (0.5% w / v) composition at a concentration of 50 mg / mL even when heated to 90 ° C. I did not.

In another experiment, M ₄ N is the total concentration tested in HP-β-CD (50% w / v) and HPMC (0.5% w / v) composition without heating, ie 1 mg. Suspensions were formed at / mL, 10 mg / mL, 20 mg / mL and 50 mg / mL. These suspensions were stable at room temperature for over 7 days.

The results in Table 15 also indicate that M ₄ N was soluble at 1 mg / mL in the EC formulation without application of heat and was stable for more than 3 days at room temperature. M ₄ N was soluble at a concentration of 10 mg / mL at 40 ° C. and remained in solution after cooling, and the solution was stable at room temperature for over 3 days. M ₄ N was soluble in the EC formulation at a concentration of 20 mg / mL at 60 ° C. and remained in solution after cooling, and the solution was stable at room temperature for over 3 days. M ₄ N at a concentration of 30 mg / mL was soluble in the EC formulation at 60 ° C., but could not remain in solution by cooling. Higher concentrations of M ₄ N, such as the 50 mg / mL or 100 mg / mL levels, were soluble in EC formulations at 90 ° C., but could not remain in solution upon cooling.

M ₄ N also formed a suspension in 1% low viscosity CMC at levels of 10 mg / mL and 20 mg / mL. These suspensions were stable at room temperature for less than 2 days.

Example 5 FIG. Solubility of M ₄ N in water-insoluble lipids and water-soluble organic solvents

A 10 mL solution of M ₄ N at a concentration of about 50 mg / mL in sesame oil was prepared as follows. About 10 mL of sesame oil was added to a glass beaker with a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 500 mg of M ₄ N was slowly added to sesame oil in the center of the beaker using a spatula to prevent M ₄ N from attaching to the beaker wall. The M ₄ N / sesame oil mixture was stirred for about 2 hours or until all M ₄ N was dissolved or homogeneously suspended and no lumps were present. M ₄ N / sesame oil mixture at about 60 ° C. for about 30 minutes (or longer if a larger volume of solution is desired, eg 1 hour at 60 ° C. for 500 mL of M ₄ N / sesame oil mixture) or required In response to this, M ₄ N was completely dissolved by heating for a longer time. The M ₄ N / sesame oil mixture or the presence of undissolved M ₄ N is determined by holding the beaker against a white background and then a dark background and then probing for the presence of particulates. The solution was observed. When crystals formed, the M ₄ N / sesame oil solution was again heated at 60 ° C. for 1 hour with stirring on a high temperature magnetic plate until all the M ₄ N dissolved and returned to the solution. The final M ₄ N / sesame oil solution was stored at room temperature in the dark.

The desired volume or concentration of M ₄ N may be obtained by scaling up or down the above method, and the heating temperature may be increased or decreased to achieve dissolution of M ₄ N. Formulations containing M ₄ N in other water-insoluble lipids are similarly obtained, for example, by replacing sesame oil with corn oil, olive oil, soybean oil, peppermint oil or other solubilizers and combinations thereof in the manner described above. May be prepared. The results are shown in Table 16.

A mixture of 10 g M ₄ N at a concentration of about 200 mg / g in 95% olive oil and 5% beeswax was prepared as follows. About 7.6 mL of olive oil was added to a glass beaker with a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 2 g of M ₄ N was slowly added to olive oil in the center of the beaker using a spatula to prevent M ₄ N from sticking to the beaker wall. The M ₄ N / olive oil mixture was stirred for about 2 hours, or until all M ₄ N was dissolved or homogeneously suspended and no lumps were present. The M ₄ N / olive oil mixture is optionally about 60 minutes at 60 ° C. (or longer if a larger volume of the solution is desired, eg 1 at 60 ° C. for a 500 mL M ₄ N / olive oil mixture. M ₄ N was dissolved completely by heating for a longer time if necessary. M ₄ N / olive oil mixture to determine whether undissolved M ₄ N is present by holding the beaker against a white background and then a dark background and then exploring the presence of particulate matter Was observed. If crystals formed, the M ₄ N / olive oil solution was heated again at 60 ° C. for about 1 hour with stirring on a high temperature magnetic plate until all the M ₄ N dissolved and returned to the solution. About 400 mg of white beeswax was added to a glass beaker with a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. The beeswax was heated at about 50 ° C. for about 30 minutes (longer if more is required) or until all the beeswax has melted. The M ₄ N / olive oil solution was then added to about 400 mg of melted beeswax and stirred and heated at about 50 ° C. for about 30 minutes or until the entire M ₄ N / olive oil / beeswax mixture was dissolved or homogeneously mixed. The stir bar was removed from the beaker and the M ₄ N / olive oil / beeswax mixture was cooled. The final M ₄ N / olive oil / beeswax mixture was stored at room temperature in the dark.

The desired volume or concentration of M ₄ N may be obtained by scaling up or down the method. Formulations containing M ₄ N in other water-insoluble lipids can be obtained, for example, by replacing olive oil in the method described above with corn oil, sesame oil, soybean oil, peppermint oil, lecithin or other solubilizers and combinations thereof. And beeswax may be similarly prepared by replacing beeswax with paraffin, PEG 3350 or other curing agents and combinations thereof. If desired, a bioavailability-enhancing agent such as eugenol, cinnamaldehyde, lecithin, naringenin, naringin and piperine may be included in combination with any of the carriers. The results are shown in Table 16.

A solution of 10 mL M ₄ N at a concentration of about 60 mg / mL in 85% sesame oil and 15% Tween 20 was prepared as follows. About 8.5 mL of sesame oil was added to a glass beaker containing a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 1.5 mL of Tween 20 was slowly added to the center of the beaker. About 600 mg of M ₄ N was slowly added to the sesame oil / Tween 20 mixture in the center of the beaker using a spatula to prevent M ₄ N from sticking to the beaker wall. The M ₄ N / sesame oil / Tween 20 mixture was stirred for about 2 hours or until all M ₄ N was dissolved or evenly suspended and no lumps were present. The M ₄ N / sesame oil / Tween 20 mixture is about 30 minutes at 60 ° C. (or longer if a larger volume of the solution is desired, eg at 60 ° C. for a 500 mL M ₄ N / sesame oil / Tween 20 mixture. 1 hour), or by heating for a longer time if necessary, M ₄ N was completely dissolved. Whether or not undissolved M ₄ N is present is determined by holding the beaker against a white background and then a dark background and then exploring for the presence of particulate matter. M ₄ N / sesame oil / Tween 20 mixtures were observed. The final M ₄ N / sesame oil / Tween 20 solution was stored at room temperature in the dark. If crystals form during storage, the M ₄ N / sesame oil / Tween 20 solution can be heated again to 60 ° C. with stirring on a high temperature magnetic plate until all the M ₄ N has dissolved and returned to the solution.

By scaling up or down the process, the desired volume or concentration of M ₄ N may be obtained, and the heating temperature may be increased or decreased to achieve dissolution of M ₄ N. Formulations containing M ₄ N in non-ionic surfactants, ionic surfactants, other water-insoluble lipids combined with water-soluble organic solvents can be obtained from, for example, sesame oil or Tween 20 in corn oil in the method described above. , Olive oil, soybean oil, peppermint oil, Tween 80, TPGS, lecithin, PEG 300, PEG 400, PEG 400 monolauric acid, glycerol, PVP, PG or other solubilizers and combinations thereof and prepared similarly It's okay. The results of solubility of M ₄ N in water-insoluble lipids are shown in Table 16.

Formulations containing M ₄ N in non-ionic, ionic or amphiphilic surfactants, or other water-insoluble lipids in combination with water-soluble organic solvents can be obtained from, for example, sesame oil in corn oil , Olive oil, soybean oil, peppermint oil or mineral oil, and Tween 20 is replaced with Tween 80, TPGS, lecithin, PEG 300, PEG 400, PEG 400 monolauric acid, glycerol, PVP, PG or other solubilizers and these May be prepared similarly by substituting the combination of The results of the solubility of M ₄ N in water-insoluble lipids and the stability of such compositions are shown in Table 16. Stability for a clear liquid solution herein refers to the time until it forms a crystallized precipitate in solution. A stable solution is one that is clear and does not contain particulates over a long period of time.

Table 16 shows that, for example, M ₄ N is soluble in corn oil when heated at 60 ° C. at concentrations up to 100 mg / mL, and lower concentrations of M ₄ N are cooled except at the 100 mg / mL level. Later it stays in solution, the solution is stable for more than 3 days at 1 and 10 mg / mL concentrations, less than 3 days at levels of 20, 40 and 50 mg / mL, and less than 1 day at levels of 60 mg / mL, It shows that it is stable. Furthermore, M ₄ N was soluble in olive oil at 60 ° C. at a level of 30 mg / mL, but could not remain in solution after cooling.

In sesame oil, M ₄ N was soluble at room temperature at a level of 10 mg / mL. At 60 ° C., M ₄ N was soluble to a concentration of 50 mg / mL and remained in solution after cooling. The 10 mg / mL and 20 mg / mL solutions were stable for more than 3 days at room temperature, the 30 mg / mL solution was stable for less than 3 days at room temperature, and the 50 mg / mL solution was stable for less than 1 day at room temperature.

In peppermint oil, _{M 4} N was soluble in the range of 1mg / mL~125mg / mL. At concentrations up to 20 mg / mL, M ₄ N was soluble without heating. These compositions were stable at room temperature for more than 3 days. M ₄ N was soluble at higher concentrations up to 125 mg / mL level by heating at 40 ° C. and remained in solution after cooling. Of these higher concentrations of M ₄ N in peppermint oil, the 40 mg / mL composition was stable at room temperature for over 3 days.

M ₄ N was also soluble in soybean oil at concentrations up to 50 mg / mL by heating at 60 ° C. Of these, only the 10 mg / mL concentration remained in solution upon cooling and the solution remained stable for over 7 days.

M ₄ N was also soluble in mineral oil at concentrations up to 200 mg / mL when heated at 60 ° C. The 10 mg / mL and 50 mg / mL compositions remained in solution upon cooling. Of these, the 10 mg / mL solution was stable for more than 7 days at room temperature.

In a combination of 50% peppermint oil and 50% PEG 300, M ₄ N was soluble to 125 mg / mL when heated at 35 ° C. At levels between 40 mg / mL and 60 mg / mL, M ₄ N remained in solution after cooling and was stable for more than 7 days at room temperature. In the combination of 60% peppermint oil and 40% PEG 300, M ₄ N was soluble at 60 mg / mL when heated at 40 ° C. and remained in solution when cooled. This composition was stable for more than 3 days at room temperature.

When peppermint oil was combined with PEG 400 at 50% each, M ₄ N was soluble to 125 mg / mL when heated at 40 ° C. At 40 mg / mL and 60 mg / mL M ₄ N concentrations, the compound remained in solution upon cooling and the composition was stable for more than 7 days at room temperature. In the combination of 60% peppermint oil and 40% PEG 400, M ₄ N was soluble at 60 mg / mL when heated at 40 ° C.

In a combination of 50% peppermint oil and 50% Tween 20, M ₄ N was soluble up to 125 mg / mL tested by heating at 40 ° C. Of these, M ₄ N remained in solution after cooling at concentrations of 40 mg / mL and 60 mg / mL.

In other combinations such as 50% peppermint oil and sesame oil each, M ₄ N was soluble up to the 60 mg / mL concentration tested. M ₄ N was soluble at 20 mg / mL at room temperature and the composition was stable for more than 3 days at room temperature. The 40 mg / mL and 60 mg / mL solutions of M ₄ N remained in solution upon cooling and were stable at room temperature for over 3 days.

In yet another combination containing 33% peppermint oil, 33% Tween 20, and 33% PEG 400, M ₄ N was soluble at 60 mg / mL when heated at 40 ° C. and remained in solution when cooled. These compositions were stable for over 3 days.

Table 16 also shows, for example, that M ₄ N can be solubilized in soybean oil and can become a waxy solid when combined with beeswax. Furthermore, M ₄ N can also be solubilized in olive oil and beeswax can be added to convert the composition into a waxy solid.

Table 17 shows the results obtained for the solubility of M ₄ N in the water-soluble organic solvents EtOH, PG, PEG 300, PEG 400, PEG 400 monolauric acid, glycerol, PVP and certain combinations thereof.

Example 6 Solubility of M ₄ N in nonionic surfactants

  In preparing a 10 mL solution of 20% TPGS (w / v) for use as a solubilizer and / or excipient, 8 mL of WFI was charged into a glass beaker with a stir bar. The beaker was placed on a high temperature magnetic plate and the stir bar was set to stir at medium speed. The WFI was heated at about 95 ° C. for 5 minutes. 2 g TPGS was added to another glass beaker and heated at 40 ° C. for 15 minutes or until all the TPGS was melted. The melted TPGS was slowly added to the near-boiling WFI while using a spatula to direct the TPGS to the center of the beaker, thereby preventing the TPGS from sticking to the beaker wall surface. The 20% TPGS solution was stirred for about 24 hours or until it was visually determined that the TPGS was completely dissolved. The final solution was then stored at room temperature in the dark.

  The desired volume or concentration of the TPGS solution may be obtained by scaling up or down this method of preparing TPGS. Other TPGS solutions are prepared in the same manner as described above in combination with other nonionic surfactants, ionic surfactants, amphiphilic surfactants, water-soluble organic solvents or other solubilizers. It's okay. The results are shown in Table 18.

A 10 mL solution of M ₄ N at a concentration of about 60 mg / mL in Tween 20 was prepared as follows. About 10 mL of Tween 20 was added to a glass beaker with a stir bar. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. About 600 mg of M ₄ N was slowly added to Tween 20 in the center of the beaker using a spatula to prevent M ₄ N from sticking to the beaker wall. The M ₄ N / Tween 20 mixture was stirred for 2 hours or until all M ₄ N was dissolved or uniformly suspended and no lumps were present. M ₄ N / Tween 20 mixture at about 60 ° C. for about 30 minutes (or longer if a larger volume of solution is desired, eg 1 hour at 60 ° C. for 500 mL of M ₄ N / Tween 20 mixture) or required In response to this, M ₄ N was completely dissolved by heating for a longer time. M ₄ N / Tween 20 mixture for the presence of undissolved M ₄ N by holding the beaker against a white background and then a dark background and then exploring the presence of particulate matter Or the solution was observed. The final M ₄ N / Tween 20 solution was stored at room temperature in the dark. If crystals formed during storage, the M ₄ N / Tween 20 solution was again heated at 60 ° C. for about 1 hour with stirring on a high temperature magnetic plate until all the M ₄ N dissolved and returned to the solution. .

By scaling up or down the method, the desired volume or concentration of M ₄ N may be obtained, and the heating temperature may be increased or decreased to achieve dissolution of M ₄ N. Formulations containing M ₄ N in other non-ionic surfactants, ionic surfactants or amphiphilic surfactants can be prepared using, for example, Tween 20 in the method described above as Tween 80 or other solubilizing agents and Similar preparations may be made by substituting these combinations. Table 18 shows the solubility results of M ₄ N in Tween 20, Tween 80, and combinations of Tween 20 and PEG 400.

Table 18 shows that M ₄ N is soluble in Tween 20 and Tween 80 at a concentration of 1 mg / mL at room temperature. Tween 20 solution of _M 4 N (hereinafter, _"M 4 N / Tween 20" referred to) is stable at room temperature over 7 days, Tween 80 solution of _M 4 N (hereinafter, _"M 4 N / Tween 80 ”) was stable for more than 3 days of observation. Higher concentrations of M ₄ N were also soluble in Tween 20 or Tween 80 at 50 ° C. Furthermore, M ₄ N remained in solution after cooling at concentrations up to 60 mg / mL in Tween 20 and up to 50 mg / mL in Tween 80, but 80 mg / mL and 100 mg / mL in Tween 20 The mL level became insoluble upon cooling. The 10 mg / mL and 20 mg / mL M ₄ N / Tween 20 solutions were observed to be stable at room temperature for over 7 days. The 40 mg / mL and 80 mg / mL M ₄ N / Tween 20 solutions were observed to be stable at room temperature for less than 3 days. For the M ₄ N / Tween 80 solution, the 10 mg / mL solution was observed to be stable at room temperature for over 3 days, while the 50 mg / mL solution was stable at room temperature for less than 1 day.

The results also show that M ₄ N was stable up to a 60 mg / mL concentration of M ₄ N tested when heated at 65 ° C. in the combination of 50% Tween 20 and 50% PEG 400. M ₄ N remained in solution in these formulations upon cooling, and the solution was stable at room temperature for over 3 days.

  The combination of PEG 400 and Tween 20 was tested at various heating temperatures, length of time the heat was applied, cooling method and time, and drug concentration added.

Preparation of a 10 mL solution was initiated by adding 10 mL of glycerol monooleic acid (hereinafter referred to as “Glymo”) to a glass beaker. The beaker was placed on a magnetic plate and the stir bar was set to stir at medium speed. The drug was dissolved by heating it at 60 ° C. for 30 minutes. The mixture was observed for the presence of undissolved M ₄ N by holding the beaker against a white background and then a dark background and then probing for the presence of particulates. The Glymo mixture was cooled to room temperature with heating, and the mixture was made into a suspension. This was stored at room temperature in the dark. The suspension remained stable for 2 weeks and then began to separate. Recombine by shaking.

  Example 7

47.5% (v / v) Tween 20, 47.5% (v / v) PEG 400, 2.5% (w / v) PEG 3350, 2.5% (w / v) beeswax of Example 6. The melting point (see Table 18) was measured for several concentrations of M ₄ N as follows (Table 19).

Example 8 FIG. Lyophilized preparation containing M ₄ N in HP-β-CD

A 120 mg M ₄ N lyophilized powder at a concentration of about 185 mg / g (w / w) in HP-β-CD was prepared as follows. Equimolar amounts of HP-β-CD and M ₄ N were used to increase the complex formation ratio between HP-β-CD and M ₄ N. About 98 mg of HP-β-CD and about 22.2 mg of M ₄ N were mixed in a 1.5 mL polypropylene tube. About 0.2 mL of WFI is added to the mixture in a polypropylene test tube containing the HP-β-CD / M ₄ N powder mixture and spun for 1 minute to mix HP-β-CD / M ₄ N in water. A suspension of was formed. The HP-β-CD / M ₄ N suspension was frozen at −20 ° C. for 24 hours. Next, the HP-β-CD / M ₄ N suspension was centrifuged at 1400 rpm under vacuum at 60 ° C. for about 2 hours to remove all water from the suspension. The dry powder of HP-β-CD / M ₄ N complex weighed about 120 mg. This HP-β-CD / M ₄ N powder complex was then dissolved or resuspended in water or other solubilizer. The final M ₄ N / HP-β-CD powder complex was stored at room temperature in the dark. The desired volume or concentration of M ₄ N may be obtained by scaling up or down the method. Formulations containing M ₄ N in other cyclodextrins may be similarly prepared, for example, by replacing HP-β-CD in the above method with other cyclodextrins. The results shown in Table 1 show that HP-β-CD after lyophilization of a HP-β-CD / M ₄ N suspension consisting of about 81.5% HP-β-CD and 18.5% M ₄ N. This shows that a powder composite of / M ₄ N was obtained.

400 mg of M ₄ N lyophilized powder at a concentration of about 150 mg / g (w / w) in HP-β-CD / HPMC was prepared as follows. Approximately 1 mL of 50% HP-β-CD / 0.5% HPMC solution, prepared as described above, was added to a 1.5 mL volume polypropylene test tube. About 60 mg of M ₄ N is added to a polypropylene tube containing the HP-β-CD / HPMC suspension, and the HP-β-CD / HPMC / M ₄ N suspension is added by rotary mixing for 1 minute. Prepared. The HP-β-CD / HPMC / M ₄ N suspension was frozen at −20 ° C. for 24 hours. The HP-β-CD / HPMC / M ₄ N suspension was then centrifuged at 1400 rpm under vacuum at 60 ° C. for about 5 hours to remove all water from the suspension. The dry powder of HP-β-CD / HPMC / M ₄ N complex weighed about 400 mg. This HP-β-CD / HPMC / M ₄ N powder complex was then dissolved or resuspended in water or other solubilizer. The final M ₄ N / HP-β-CD / HPMC powder complex was stored at room temperature in the dark. By scaling up or down the method, the desired volume or concentration of M ₄ N may be obtained. Formulations containing M ₄ N in other cyclodextrin / cellulose solutions can be obtained, for example, by replacing HP-β-CD with other cyclodextrins in the above method or replacing HPMC with other modified celluloses. May be similarly prepared. The results shown in Table 15 show that HP-β-CD / HPMC / M ₄ N suspension consisting of about 84% HP-β-CD, 1% HPMC and 15% M ₄ N after lyophilization It shows that a powder composite of β-CD / HPMC / M ₄ N was obtained.

Example 9 Absorption of M ₄ N in Sprague Dawley rats during oral treatment of liquid formulations.

Ten groups of male rats (n = 5 per group) (age = 8-10 weeks) were exposed to M ₄ N in vehicle by oral gavage to achieve a single dose of 500 mg / kg. Oral liquid excipients were examined. The animals were fasted overnight before dosing. Excipients / formulations tested were as described in Table 19, formulations prepared as described above: (a) HP-β-CD + HPMC; (b) HP-β-CD + CMC; (c) TPGS; (d) TPGS + PEG 400 (E) Tween 20; (f) PEG 400 + Tween 20; (g) PEG 400 + Tween 20 + peppermint oil; (h) peppermint oil + PEG 400; (i) peppermint oil + Tween 20; (j) peppermint oil + sesame oil. . Blood was collected from each animal via the jugular vein before administration and at 0.5, 1, 2, and 3 hours after administration. The concentration of M ₄ N in serum is measured by LC / MS / MS, where LC means liquid chromatography and MS means mass spectrometry. This method is separated by LC and then analyzed for M ₄ N detected and quantified by two consecutive rounds of MS.

As shown in FIG. 3 and Table 21, the absorption of M ₄ N varied between excipient formulations. Absorption is highest at PEG 400 + Tween 20, followed by PEG 400 + Tween 20 + peppermint oil, followed by peppermint oil + Tween 20, followed by peppermint oil + PEG 400, followed by peppermint Oil + sesame oil, next high or equivalent is Tween 20, next high or equivalent is HP-β-CD + CMC, then high is HP-β-CD + HPMC, then high is TPGS, then The high value was TPGS + PEG 400.

  In Table 21, the following four formulations are shown as stable suspensions: HP-β-CD + HPMC, HP-β-CD + CMC, TPGS and TPGS + PEG 400. The criterion used to designate these suspensions as “stable” was that the suspension showed no precipitation of its contents (the component did not settle from the suspension to the bottom of the beaker). . The other formulations in the rat study were clear liquid solutions, not suspensions, and did not have any floating components.

Example 10 Absorption of M ₄ N in beagle dogs by oral administration

5 groups of dogs (n = 2 per group, 1 male and 1 female) (age = about 6-9 months) exposed to M ₄ N in vehicle to a single 100 mg / kg dose, The optimal oral solid excipient was investigated. A composition of M ₄ N in excipient was administered orally after encapsulating in size 12 hard gelatin capsules. The animals were fasted overnight before dosing. The excipients / formulations tested are as shown in Table 19, and (a) encapsulate M ₄ N without excipients; (b) HP-β-CD; (c) TPGS; (d ) Soybean oil + beeswax; and (e) Olive oil + beeswax. Blood was collected from each animal via the jugular vein before dosing and at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours and 8 hours after administration. The concentration of M ₄ N in serum was measured by liquid chromatography (LC / MS / MS) combined with tandem mass spectrometry.

As shown in FIG. 4, the absorption of M ₄ N varied depending on the excipient / formulation. Absorption is highest in the formulation containing olive oil + beeswax, followed by the highest value containing HP-β-CD, the next highest value containing TPGS, and then the highest value soybean oil + beeswax The next highest value was M ₄ N without excipients.

While the above examples have been described using M ₄ N, other NDGA derivatives or other catecholbutanes may be substituted with appropriate adjustments in weight and volume to achieve the appropriate concentration.

Example 11-1 . Sample collection for measurement of pharmacokinetics of micronized and non-micronized M4N after oral administration to dogs

The purpose of this study was to evaluate the pharmacokinetic profile of M ₄ N when administered in micronized or non-micronized form via oral gavage in beagle dogs. Test articles were prepared in glycerol monostearic acid at 60 mg / mL. The test plan is summarized in Table 22.

  Blood (approximately 2 mL) is monoject free of anticoagulant before and after administration at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours. It was collected from the jugular vein in a 2 mL red top tube.

Absorption of M ₄ N occurred after administration of both formulations. Average pharmacokinetic parameters are as shown in Table 23, where C _max is the maximum concentration of absorbed M ₄ N and AUC is the area under the curve for the total absorbed M ₄ N. . Absorption was higher after administration of micronized M ₄ N compared to non-micronized M ₄ N. However, the concentration curves tended to be more consistent between animals after administration of non-micronized M ₄ N (FIGS. 5A and 5B on the non-logarithmic and logarithmic scales, respectively, show non-micronized M ₄ N. Absorption is shown; and FIGS. 6A and 6B on the non-logarithmic and logarithmic scales, respectively, show the absorption of micronized M ₄ N).

Example 11-2 . Comparison of oral feeding / fasting and IV / oral pharmacokinetics of two formulations of M4N in beagle dogs

The purpose of this study was serum achieved after a single 75 mg / kg dose of pre-formulated M ₄ N administered orally (by gavage or capsule) or IV in the fed or fasted state It was to evaluate the medium concentration. Three males and two females each had EM-1421 in glycerol monooleic acid (hereinafter referred to as “Glymo”), M ₄ N in Tween 20 / PEG 400 / naringin (hereinafter referred to as “TPN”), or 50 M ₄ N in 20% hydroxypropyl-β-cyclodextrin (HPβCD) (hereinafter referred to as “CPE”) in% PEG 300 was administered. Glymo and TPN were also administered in non-concentrated (60 mg / mL) or concentrated (300 mg / mL) state as shown in the legends of FIGS. 7B and 8B.

  All animals survived throughout the study. Clinical observations included diarrhea, vomiting and ataxia (ataxia occurred in one female after IV administration and persisted for about 1 hour).

The serum concentration of M ₄ N varied greatly depending on the formulation and condition. Table 24 summarizes these findings. Serum concentration was lowest after administration of concentrated Glymo or TPN. C _max was highest after TPN administration (non-concentrated, fasted state) (Table 24 and FIGS. 7A and 8A, non-logarithmic scale, M ₄ N serum concentration administered orally to male and female dogs, respectively) Showing). AUC peaked after Glymo administration (non-concentrated, fasted state) (Table 24 and FIGS. 7B and 8B, log scale, showing serum concentration of M ₄ N administered orally to male and female dogs, respectively) . The AUC achieved after oral administration of Glymo (non-concentrated, fasted state) was 35% (male) and 47% (female) of AUC achieved after IV administration, assuming IV administration was 100%. (Table 24).

Example 12 Pharmacokinetic study of repeated administration of M ₄ N (oral gavage) in rats

The purpose of this study was to provide information on the pharmacokinetics of a 500 mg / kg dose of M ₄ N in 10 excipient formulations. Fifty Crl: (CD) SD male and female rats were assigned to 10 dose groups of 5 males and 5 females per group. Samples with the following carriers administered to the following subject groups were prepared (Table 25).

Three types of M ₄ N formulations were orally administered by gavage. M ₄ N at a concentration of 60 mg / mL was administered at a dose of 8.33 mL / kg based on recent body weight. Rats were given an initial dose (first day of study) after an overnight fast, followed by a 72-hour withdrawal (recovery) period. On study day 5 (DS5), the second dose of test article was administered to non-fasted rats followed by a one week rest (recovery) period. In DS6, rats were placed on a high fat diet (containing about 10% fat compared to about 5% in the standard diet). In DS12, non-fasted rats were given a third dose. Rats were observed during habituation at least twice daily for survival and weekly for clinical observations and general appearance. Rats are also clinically observed at the appropriate time interval of the first 4 hours before and after dosing, and at the end of the normal work day on the first day of dosing and at about 2 hours the day after dosing. Tested for death. These observations were recorded once a day during the non-medication day and during the period following the dosing period. Body weight was recorded at least weekly during the acclimation period, daily during dosing, and as terminal body weight at sacrifice. Food intake values were recorded weekly during the dosing period and at slaughter (residual feed volume). On day 1, day 5 and day 12 of the study, blood samples (approximately 0.5 mL each) were taken via the lateral tail vein of each rat assigned to the study. Each blood collection was recorded as raw data. Blood samples were collected from each rat on day 1, 2 and 12 of the study at the following time points: before dosing, 15 minutes after dosing and 1 and 4 hours after dosing. Samples were transferred to serum separation tubes and centrifuged. The obtained serum was transferred to a polypropylene test tube labeled with the protocol number, tester's test number, rat number, group number, dosage, test date, collection interval, collection date, species, generation and storage conditions. All samples were frozen immediately on dry ice and kept frozen (−68 to −78 ° C.) until shipped for analysis.

All rats survived until the scheduled sacrifice. Urine stained abdominal body hair was observed in 4 out of 5 female rats (Group IX) administered PEG 400 / Tween 20 / Peppermint oil. This symptom occurred in DS 2-3, but did not persist. Soft or liquid feces was the only clinical observation that occurred in at least some animals in each group except for rats administered PEG 400 / Tween 20 / Peppermint oil (Group IX). All groups showed average weight gain during the study period. When fed a high fat diet, the weight gain of male and female rats in DS6-13 was always lower than in DS1-6 when fed a standard diet. Body weight gain generally excludes DS6-13 female rats in PEG 400 / Tween 20 and PEG 400 / Tween 20 / PEG 3350 / Beeswax dose groups (Groups I and X, respectively) that showed low weight gain It was comparable between groups. Absolute and relative food intake values were similar between groups throughout the study. No gross lesions related to the test product were observed at necropsy. Absorption of M ₄ N was observed in all solvents. Blood levels in rats on a high fat diet were always higher than those observed after standard diet or fasting. After administration of a high fat diet, the highest absorption was observed in the PEG 400 / Tween 20 / naringin group. The highest absorption was observed in the PEG 400 / Tween 20 / Naringin group after standard diet administration. After fasting, the highest absorption was observed in the PEG 400 / Tween 20 / Peppermint oil group. Maximum exposure levels are generally reached within 1 hour after dosing, with the exception that glycerol monooleic acid formulations continue to rise at 4 hours after dosing.

In conclusion, all test article formulations were tolerated, there were no deaths, and all groups showed weight gain. Soft and liquid feces were the most common clinical observations, but there was no effect on food intake in any solvent. M ₄ N blood concentration was always high when rats were fed a high fat diet. The highest exposure was observed within 1 hour after dosing except for glycerol monooleate.

Example 13 Human phase 0 ternary crossover microdose pharmacokinetic study of ¹⁴ C-labeled M ₄ N in 8 healthy male subjects

This study is for M ₄ when administered to humans as a sub-therapeutic dose as either a single oral dose or a single intravenous dose (use AC in Table 26, respectively) under fed and fasted conditions. Designed to evaluate N absorption. The study was a three-phase crossover study design in a target population of healthy male subjects and consisted of three study periods consisting of approximately 35 hours each separated by a minimum period of at least 7 days between dosing. During the course of each study period, blood samples for pharmacokinetics were collected at specific time points after dosing and urine was collected at predetermined time intervals. Subjects were discharged after completing certain treatments in the study 24 hours after dosing.

In this study, M ₄ N was administered to humans in an amount of 100 μg. M ₄ N was slightly labeled with ¹⁴ C (3.3 kBq per 100 μg) and administered to healthy volunteers. Each oral dose of M ₄ N is a gelatin capsule of size 0, consisted of glycerol monooleate of _{M 4} N and 376.8mg was ¹⁴ C labeled 0.1 mg. A single intravenous infusion of M ₄ N consists of 0.1 mg / mL ¹⁴ C-labeled M ₄ N, 30% (w / v) HP-β-CD and 1 mL diluted with water for instantaneous bulk injection. It consisted of 25% (v / v) PEG. After collecting blood and urine from each subject, the samples were analyzed for ¹⁴ C content using an accelerator mass spectrometer (AMS) to determine the maximum concentration of M ₄ N at T _max (C _max ), time point at which the test was collected The total area under the curve (AUC) representing all absorption of M ₄ N in (AUC _0-t ) and all (AUC _0-∞ ), the terminal half-life (T _1/2 ) of each sample, and M The relative and total bioavailability (F _rel and F) of oral administration of M ₄ N as compared to ₄ N IV dosing was determined. Mean ± SD values of pharmacokinetic parameters for ¹⁴ C are shown in Table 26. By baseline levels of M 4 _N is to correct for M _{4 N} remaining subject after a period of between doses, the level of M _{4 N} for any of the following dosing was prevented exaggerated.

Regarding the oral dose, the C _max value for total ¹⁴ C is greater after oral administration after overnight fasting after oral administration of 100 μg of ¹⁴ C-labeled M ₄ N after high fat breakfast (C _max = 5.94 ± 1.33 pmol / L). The value was lower than (C _max = 9.29 ± 1.40 pmol / L). T _max was more delayed in feeding than in fasting. In fed subjects, the time of T _max , ie C _max , is highly variable, ranging from 1.5 to 24 hours after dosing, and in fasting subjects, T _max is generally post-dosing. Happened in 1 hour (range 0.50-2.00 hours). AUC _0-t values were generally lower in fed subjects than in fasted subjects.

After intravenous dosing, the highest concentration (C _max = 10.31 ± 1.77 pmol / L) was observed as expected at the first sampling (0.08 hours after dosing) in 8 of 10 subjects. In 2 subjects, the T _max was 0.17 hours after dosing. AUC _0-t values for total ¹⁴ C plasma concentrations were slightly lower after single oral dosing in fed subjects than after intravenous dosing. Conversely, the corresponding AUC _0-t values were slightly higher after a single oral dose in fasted subjects than after intravenous administration.

  The test formulation was well tolerated for both oral and intravenous administration. No serious or severe adverse events were observed, and none of the subjects were interrupted by adverse events related to study administration. No clinically significant changes in vital signs or ECG were observed.

In conclusion for this study, the apparent absorption of M ₄ N was very high after oral administration in the fed and fasted state. In the presence of food intake, the rate and extent of absorption was low compared to the fasted state, and the time at _Cmax was prolonged in the administration at food intake. These conclusions are based on the assumption that ¹⁴ C labeled M ₄ N oral doses are not degraded prior to absorption.

Example 14 Additional tests on the solubility of M ₄ N in water-soluble organic solvents

The solubility of M ₄ N in the water-soluble organic solvent combinations shown in Table 27 was evaluated for 48 hours. After 2, 24 and 48 hours of incubation at room temperature, the solubility of M ₄ N was quantified by analyzing the sample by reverse phase HPLC (“RP-HPLC”). To prepare the M ₄ N sample, 200 μL of 100 mg / mL M ₄ N dissolved in acetone was added to a 1.5 mL polypropylene micro test tube. The solvent was allowed to evaporate at room temperature for 48 hours until the sample was completely dry.

  A water miscible organic solvent formulation was prepared in a 15 mL polypropylene centrifuge tube. 10 mL of each formulation was prepared. Each solvent was added on a weight basis using its corresponding density at 25 ° C. After mixing briefly, each formulation was filtered through a 0.45 μm surfactant-free cellulose acetate (“SFCA”) filter and collected in a new 15 mL test tube. The formulation was stored at room temperature until use.

400 μL of each formulation combination (Table 27, Benz = benzyl alcohol; Crem = Cremophor EL; DMA = dimethylacetamide; T80 = Tween 80) was added to the micro test tube for a maximum solubility of 50 mg / MLM ₄ N. M ₄ N solubility was assessed by RP-HPLC during 2, 24 and 48 hours incubation at room temperature. At each time point, the solid M ₄ N present was pelleted by centrifuging the sample at 13000 rpm for 2 minutes. As shown in Table 27, more than half of the formulation conditions tested were able to solubilize M ₄ N to concentrations greater than 10 mg / ML. The solubility of M ₄ N in glycerol at 2 and 48 hours was undetectable.

Example 15. Solubility of M ₄ N in aqueous solution

Hydroxypropyl HP-β-CD or sulfobutyl ether β-cyclodextrin (SE-β-CD) (Captisol (registered trademark: hereinafter, “Captisol” is a registered trademark)), CyDex, Inc., Lenexa, The solubility of M ₄ N dissolution in an aqueous solution containing any of (KS, USA) was evaluated at room temperature as described above in Example 14 for up to 48 hours. Ten specimens of 50% solutions of HP-β-CD and SE-β-CD were prepared on a weight to volume basis. M ₄ N used in the samples was prepared as described in Example 14. 1.0 g to 5.0 g of each compound was weighed into a 10 mL volumetric flask on an analytical balance (OHAUS Analytical Plus Balance). Each sample was made up to 10 mL (qs) with water for injection (WFI). After 1 hour incubation at 40 ° C., the preparation was filtered through a 0.45 μm SFCA filter and collected into a new 15 mL test tube. The preparation was stored at room temperature until use.

As shown in Table 28, the solubility of M ₄ N in WFI, 0.9% saline, 5% glucose (DSW) was below the RP-HPLC quantitation limit throughout the course of this study. Note the increased M ₄ N solubility as a function of HP-β-CD and Captisol concentrations and time.

More than 20 formulation conditions with water miscible solvents were able to solubilize M ₄ N to concentrations greater than 10 mg / mL (greater than 10 mg / mL). The solubility of M ₄ N in WFI, 0.9% saline, D5W was below the detection limit of the RP-HPLC method. The solubility of M ₄ N increases as a function of HP-β-CD and Captisol concentrations and time.

Example 16 Solubility of M ₄ N in hydroxypropyl β-cyclodextrin

The aqueous solubility of M ₄ N at various concentrations of HP-β-CD was evaluated by the method reported by Higuchi and Connors (1965). That is, accurately measure M ₄ N, add in excess of its water solubility, and gently rotate at room temperature (˜12 rpm) with an aqueous solution of HP-β-CD with increasing concentration (0-350 mmol / L) for 48 hours. ) The M ₄ N / HP-β-CD solution was then filtered through a 0.45 μm SFCA filter and analyzed by RP-HPLC.

Although most drug / cyclodextrin complexes were thought to be inclusion complexes, cyclodextrins form complex aggregates that can dissolve drugs through non-inclusion complexes and micelle-like structures. Is also known. The phase-solubility profile did not confirm the formation of the inclusion complex, but only explained how increasing concentrations of cyclodextrin affect drug solubility. The formation of the M ₄ N / HP-β-CD complex was non-linear, but exact stoichiometric (as well as stability constant) measurements were not examined in the experiments of this example. However, it can be measured by other means such as NMR or potentiometry.

Example 17. Solubility of M ₄ N in HP-β-CD / PEG 300 buffer solution

Stability of 10 mg / mL M ₄ N (75:25, 40% HP-β-CD: prepared at a ratio of 40 mg / mL M ₄ N PEG 300) in 15 mM buffer solution at 60 ° C. Rated after.

A buffered 40% solution of HP-β-CD was prepared on a weight basis to volume. M ₄ N used in the samples was prepared as described in Example 14. 2.0 g of HP-β-CD was weighed into a 5 mL volumetric flask on an analytical balance (OHAUS Analytical Plus Balance). 1 mL of 100 mM buffer solution was added to each flask. Each sample was made up to 5 mL with WFI. After 1 hour incubation at 40 ° C., the preparation was filtered through a 0.45 μm SFCA filter into a new 15 mL test tube. The preparation was stored at room temperature until use.

750 μL of 40% HP-β-CD buffer solution was put into a 1.5 mL polypropylene microtube. 250 μL of 40 mg / mL M ₄ N PEG 300 was added to each microtube to allow 10 mg / mL solubility of M ₄ N. After gently inverting the sample tube, the pH of each solution was measured using an Orion, Model 420A pH meter. The first aliquot was taken for RP-HPLC analysis. Next, the test sample was put into a Precision 60 ° C. incubator. M ₄ N Stability was evaluated by RP-HPLC. At each time point, solid M ₄ N was pelleted by centrifuging the sample at 13000 rpm for 2 minutes.

As shown in Table 29, a slight decrease in the concentration of the various M ₄ N solutions was observed after 14 days incubation at 60 ° C. RP-HPLC data revealed no increase in sample impurities corresponding to the degree of reduction in the concentration of M 4 _N. However, pH dependent changes were observed with M ₄ N impurities, but these impurities only comprised less than 0.1% of the total peak area. Even if an apparent change in pH of the sample was observed during the incubation period, it was slight (Table 29).

A uniform decrease in stability independent of the apparent sample pH or buffer, indicated by loss of M ₄ N recovery, was observed after 14 days incubation at 60 ° C.

Stability of _{M 4} N in Example 18.11~14mg / mL of PEG 300 / HP-β-CD solution

To support manufacturing specifications, this study examined the 24-hour room temperature stability of PEG 300 / HP-β-CD combinations at various target concentrations of M ₄ N. Stock samples of M ₄ N stock solutions were prepared in 100% PEG 300 at a drug concentration of 33-56 mg / mL at 60 ° C. as follows.

After incubating at 60 ° C. for at least 2 hours, M ₄ N bulk formulations were made into 33, 44, 48, 52, 55 and 56 mg / mL in PEG 300 using the procedure described in Examples 14 and 17. Solubilized to a concentration of (w / w). Vigorous rotary mixing was required for complete solubilization of above 44 mg / mL M ₄ N. The M ₄ N stock solution was filtered through a 0.45 μm SFCA filter and used within 30 minutes of preparation. Separately, a solution of 40% (w / v) HP-β-CD was prepared in sterile WFI and filtered. A combination of 40% HP-β-CD stock and M ₄ N / PEG 300 stock was mixed in a 1.5 mL polypropylene microtube. Stability of M ₄ N was evaluated by RP-HPLC during incubation 2 and 24 hours at room temperature.

The required concentration of M ₄ N stock solution was added to 40% HP-β-CD to give the final drug and excipient concentrations shown in Table 30. Samples were rotated gently (˜12 rpm) at room temperature. At 2 and 24 hours of incubation, the samples were centrifuged at 13000 rpm for 2 minutes and 50 μL of apricot was removed for RP-HPLC analysis. Regardless of the target M ₄ N or formulation, even if a change in solubility was observed after 24 hours of incubation (Table 30).

Test samples containing 11-14 mg / mL M ₄ N formulated to a final concentration of 25% PEG 300 and 30% HP-β-CD were stable after 24 hours incubation at room temperature.

Example 19. Stability of ₄₀ mg / mL M ₄ N / PEG 300

The stability of 40 mg / mL M ₄ N dissolved in 100% PEG 300 was evaluated by incubation at 30 ° C., 45 ° C. and 60 ° C. for up to 24 hours. A stock solution of 40 mg / mL M ₄ N in PEG 300 was prepared at 60 ° C. according to the procedure described in Example 18. Thereafter, the apricot was removed and incubated at an appropriate temperature. Samples were spun at 450 rpm during the entire incubation. Visual observation and RP-HPLC data collection were performed throughout. As shown in Table 31, fine crystals were observed in the 40 mg / mL M ₄ N / PEG 300 formulation after 6 hours of incubation at 30 ° C. and became more prominent after 24 hours. Crystal formation was accompanied by the disappearance of soluble M ₄ N (Table 32). Samples of 40 mg / mL M ₄ N / PEG 300 incubated at 45 ° C. and 60 ° C. were stable after 24 hours of incubation when tested by visual observation and RP-HPLC analysis (Tables 31 and 32). No change was observed in the amount or type of impurity peaks at any incubation temperature.

After 6 hours incubation at 30 ° C., microcrystals were observed in the 40 mg / mL M ₄ N / PEG 300 formulation. After 24 hours, more crystals were observed, and, as measured by RP-HPLC analysis,> 5% of the soluble _{M 4} N had disappeared.

A 40 mg / mL M ₄ N / PEG 300 sample incubated at 45 ° C. and 60 ° C. was stable after 24 hours of incubation as assessed by visual observation and RP-HPLC.

  Those skilled in the art will appreciate that the above-described embodiments can be modified without departing from the broad inventive concept. Accordingly, the invention is not limited to the specific embodiments disclosed, but is intended to embrace alterations within the spirit and scope of the invention as defined by the appended claims.

References Ansel, HC et al. (2004) Pharmaceutical Dosage Forms and Drug Delivery Systems eds., 8th ed., Lippincott Williams & Wilkins.
Garg, S. et al. (2001) Compendium of Pharmaceutical Excipients for Vaginal Formulations. Pharmaceutical Technol. Drug Delivery. Sept. 1, 2001, pp.14-24.
Gennaro, AR (2003) Remington: The Science and Practice of Pharmacy, 20th ed., With Facts and Comparisons: DrugfactsPlus. Lippincott Williams & Williams.
Higuchi T and Connors KA (1965) "Phase solubility techniques", Adv Anal Chem Instr. 4, 117-212.
Hwu, JR et al. (1998) Antiviral activities of methylated nordihydroguaiaretic acids. 1. Synthesis, structure identification, and inhibition of Tat-regulated HIV transactivation. J. Med. Chem. 41: 2994-3000.
McDonald, RW et al. (2001) Synthesis and anticancer activity of nordihydroguaiaretic acid (NDGA) and analogues. Anti-Cancer Drug Design 16: 261-270.
Rowe, RC et al. (2003) Handbook of Pharmaceutical Excipients. 4th edition. Pharmaceutical Press and American Pharmaceutical Association.

M 4 _N indicates C-33A cells lines and results of the cell proliferation tests were performed on HeLa cells lines after treatment, Figure 1A, M 4 _N M ₄ N in which exceeds the number of existing cells in the absence of treatment FIG. 5 is a graph showing the ratio of the number of cells present after treatment, where M ₄ N was provided in an amount ranging from 0 μM to 80 μM in a DMSO formulation. M 4 _N indicates C-33A cells lines and results of the cell proliferation tests were performed on HeLa cells lines after treatment, Figure 1B after M _{4 N} treatment exceeds the number of cells present in the M _{4 N} treated absence FIG. 5 is a graph showing the ratio of the number of cells present, where M ₄ N was provided in an amount ranging from 0 μM to 80 μM in an HP-β-CD / PEG formulation (“CPE” formulation). FIG. 6 is a graph showing measurement of cell death based on the ratio of dead cells for C-33A cells and HeLa cells in the absence or presence of various concentrations of M ₄ N in DMSO formulations, where the M ₄ N concentration is 0 μM. The range was ˜80 μM. FIG. 7 is a graph showing cell death measurements based on the ratio of dead cells for C-33A cells and HeLa cells in the absence or presence of various concentrations of M ₄ N in HP-β-CD / PEG formulations; The M ₄ N concentration was in the range of 0 μM to 80 μM. Sprague at 0.5 h, 1 h, 2 h and 3 h after oral administration of a single 500 mg / kg dose of M ₄ N in various liquid solubilizers and / or excipients (“carrier”) FIG. 6 is a histogram showing M ₄ N absorption in Dawley rats, with M ₄ N present at a concentration of about 60 mg / mL in all carriers. The carrier is (a) HP-β-CD (500 mg / mL) + HPMC (5 mg / mL); (b) HP-β-CD (500 mg / mL) + CMC (5 mg / mL); (c) TPGS (200 mg / mL) (D) TPGS (100 mg / mL) + PEG 400 (50% v / v); (e) Tween 20; (f) PEG 400 (50% v / v) + Tween 20 (50% v / v); g) PEG 400 (33% v / v) + Tween 20 (33% v / v) + peppermint oil (33%); (h) Peppermint oil (50%) + PEG 400 (50% v / v); (h) Peppermint oil (50%) + Tween 20 (50% v / v); (i) Peppermint oil (50%) + sesame oil (50%). Powder form or various solid carriers, ie (a) M ₄ N powder; (b) lyophilized HP-β-CD (81%) + M ₄ N (185 mg / g); (c) TPGS (20%) + M ₄ N (133 mg / g); (d) soybean oil (95%) + beeswax (5%) + M ₄ N (200 mg / g); and (d) olive oil (95%) + beeswax (5%) + M ₄ Beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours and 8 hours after a single oral dose of 100 mg / kg of N (200 mg / g) of M ₄ N graph showing absorption of _{M 4} N by. Glycerol monooleic acid by beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours after a single oral dose of 100 mg / kg graph showing the absorption of micronized M _{4 N} on the non-log scale - non in. Glycerol monooleic acid by beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours after a single oral dose of 100 mg / kg A graph showing the absorption of non-micronized M ₄ N in a logarithmic scale. Glycerol monooleic acid by beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours after a single oral dose of 100 mg / kg graph showing the absorption of micronized M _{4 N} in the non-logarithmic on the scale. Glycerol monooleic acid by beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours after a single oral dose of 100 mg / kg graph showing the absorption of micronized M _{4 N} in on the logarithm of the scale. Male beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours and 36 hours after a single oral dose of 75 mg / kg. Is a graph showing data on a non-logarithmic scale of serum concentration of M ₄ N in various carriers at various concentrations according to the abbreviation of the carrier used, the concentration of M ₄ N administered, and fasting or The designation of which dog in the fed state was administered was most clearly shown in FIG. 7B. Male beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours and 36 hours after a single oral dose of 75 mg / kg. Is a graph showing logarithmic data on the logarithm of the serum concentration of M ₄ N in various carriers at various concentrations, according to the abbreviation of the carrier used, the concentration of M ₄ N administered, and the fasting or feeding The designation of which dog in the dietary state was administered was most clearly shown. Female beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours and 36 hours after a single oral dose of 75 mg / kg Is a graph showing data on a non-logarithmic scale of serum concentration of M ₄ N in various carriers at various concentrations according to the abbreviation of the carrier used, the concentration of M ₄ N administered, and fasting or The designation of which dog in the fed state was administered was most clearly shown in FIG. 8B. Female beagle dogs at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours and 36 hours after a single oral dose of 75 mg / kg Is a graph showing logarithmic data on the logarithm of the serum concentration of M ₄ N in various carriers at various concentrations, according to the abbreviation of the carrier used, the concentration of M ₄ N administered, and the fasting or feeding The designation of which dog in the dietary state was administered was most clearly shown.

Claims (70)

A composition for oral administration to an animal comprising an active pharmaceutical ingredient and a pharmaceutically acceptable carrier, wherein the active pharmaceutical ingredient comprises a tetra -O- methyl NDGA, and the carrier is cyclodextrin And (a) a water-soluble organic solvent other than DMSO, provided that the water-soluble organic solvent is propylene glycol, the propylene glycol is in the absence of white petrolatum, in the absence of xanthan gum, and at least one of glycerin or glycine When the water-soluble organic solvent is polyethylene glycol, the polyethylene glycol is present in the absence of ascorbic acid or butylated hydroxytoluene, and the polyethylene glycol is polyethylene glycol 400. Polyethylene glycol 400 is poly Those present in the absence of a Chi glycol 8000; (b) ionic, non-ionic or amphiphilic surfactant, however when the surfactant is a nonionic surfactant the nonionic surfactant Present in the absence of xanthan gum; ( c ) modified cellulose; ( d ) a water-insoluble lipid, if the water-insoluble lipid is castor oil, the castor oil is present in the absence of beeswax or carnauba wax And a composition comprising at least one solubilizer or excipient selected from the group consisting of any combination of (a) to ( d ). 2. The composition of claim 1 comprising 0.1 mg to 200 mg of the active pharmaceutical ingredient. 3. The composition of claim 2, comprising 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 100 mg or 200 mg of the active pharmaceutical agent. The active pharmaceutical ingredients, 1 mg / mL~ 200 mg / mL, or 1 mg / g~ 250 mg / g The composition of claim 1 which is present in a concentration of. The active pharmaceutical ingredient is 1 mg / mL, 2 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 20 mg / mL mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL 5. A composition according to claim 4 present in a concentration of mL or 175 mg / mL. The active pharmaceutical ingredient is 20 mg / g, 50 mg / g, 75 mg / g, 100 mg / g, 120 mg / g, 130 mg / g, 140 mg / g, 150 mg / g, 175 mg. The composition of claim 4 present in a concentration of / g or 200 mg / g.   The composition according to claim 1, wherein the water-soluble organic solvent is selected from the group consisting of polypropylene glycol, polyethylene glycol, polyvinyl pyrrolidone, ethyl alcohol, benzyl alcohol and dimethylacetamide.   The composition of claim 1, wherein the carrier comprises polyethylene glycol. 9. The composition of claim 8, wherein the polyethylene glycol is present at a concentration of 5 % (v / v) to 100 % (v / v). The composition according to claim 9, wherein the polyethylene glycol is present at a concentration of 20 % (v / v) to 80 % (v / v). 11. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 50 % (v / v). 11. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 40 % (v / v). 11. The composition of claim 10, wherein the polyethylene glycol is present at a concentration of 33 % (v / v).   The composition of claim 8, wherein the polyethylene glycol is PEG 300. The PEG 300 is present at a concentration of 10 % (v / v), 20 % (v / v), 30 % (v / v), 40 % (v / v) or 50 % (v / v) Item 15. The composition according to Item 14.   The composition of claim 8, wherein the polyethylene glycol is PEG 400. The PEG 400 is present at a concentration of 10 % (v / v), 20 % (v / v), 30 % (v / v), 40 % (v / v) or 50 % (v / v) Item 17. The composition according to Item 16.   9. The composition of claim 8, wherein the polyethylene glycol is PEG 400 monolauric acid. 19. The composition of claim 18, wherein the PEG 400 monolauric acid is present at a concentration of 20 % (v / v) to 50 % (v / v).   The composition according to claim 1 or 8, wherein the carrier comprises unmodified cyclodextrin or modified cyclodextrin.   21. The composition of claim 20, wherein the modified cyclodextrin is selected from the group consisting of hydroxypropyl- [beta] -cyclodextrin and sulfobutyl ether [beta] -cyclodextrin. 21. The composition of claim 20, wherein the modified cyclodextrin is present at a concentration of 5 % (w / v) to 80 % (w / v). The modified cyclodextrin is 15 % (w / v), 20 % (w / v), 25 % (w / v), 30 % (w / v), 35 % (w / v), 40 % (w 23. A composition according to claim 22 present at a concentration of / v) or 50 % (w / v).   The composition of claim 1, wherein the carrier comprises a surfactant. 25. The composition of claim 24, wherein the surfactant is present at a concentration of 5 % (v / v) to 100 % (v / v). 26. The composition of claim 25, wherein the surfactant is present at a concentration of 30 % (v / v), 40 % (v / v) or 50 % (v / v).   The carrier comprises a surfactant selected from the group consisting of polysorbate, d-alpha-tocopheryl polyethylene glycol 1000 succinate, esterified fatty acid, and a reaction product of ethylene oxide and castor oil in a 35: 1 molar ratio. Item 2. The composition according to Item 1.   27. The composition of claim 26, wherein the surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80.   29. The composition of claim 28, wherein the surfactant is polysorbate 20.   The composition of claim 1 wherein the carrier comprises polyethylene glycol and a surfactant.   32. The composition of claim 30, wherein the polyethylene glycol is selected from the group consisting of PEG 300 and PEG 400.   32. The composition of claim 31, wherein the surfactant is polysorbate 20.   The composition according to claim 24, wherein the surfactant is an esterified fatty acid.   The composition of claim 1, wherein the carrier comprises modified cellulose.   35. The composition of claim 34, wherein the modified cellulose is selected from the group consisting of ethylcellulose, hydroxylpropylmethylcellulose, methylcellulose, and carboxymethylcellulose. 35. The composition of claim 34, wherein the modified cellulose is present at a concentration of 0.1 % (w / v) to 10 % (w / v).   The composition of claim 1, wherein the carrier comprises a water-insoluble lipid.   The water-insoluble lipid is selected from the group consisting of at least one of an oil, a wax and a fat emulsion, provided that the composition contains castor oil, it does not contain beeswax or carnauba wax. The composition as described.   38. The composition of claim 37, wherein the water-insoluble lipid is a fat emulsion. 40. The composition of claim 39, wherein the fat emulsion is present at a concentration of 10 % to 30 %. 40. The composition of claim 39, wherein the fat emulsion is present at a concentration of 20 %.   34. The composition of claim 33, wherein the water-insoluble lipid is an oil.   43. The composition of claim 42, wherein the oil is at least one selected from the group consisting of corn oil, olive oil, peppermint oil, soybean oil, sesame oil, mineral oil and glycerol. 43. The composition of claim 42, wherein the oil is present at a concentration of 10 % to 100 %.   43. The composition of claim 42, wherein the oil is peppermint oil.   46. The composition of claim 45, comprising sesame oil.   43. The composition of claim 42 comprising polysorbate 20.   48. The composition of claim 47, comprising polyethylene glycol.   43. The composition of claim 42, comprising polyethylene glycol.   50. The composition of claim 49, wherein the polyethylene glycol is selected from the group consisting of PEG 300 and PEG 400. The tetra-O-methyl NDGA compound is represented by the following formula (II):

[ Wherein R ₁₄ , R ₁₅ , R ₁₆ and R ₁₇ each independently represent —OCH ₃ , and R ₁₈ and R ₁₉ each independently represent —CH ₃ ] The composition of claim 1 having   The composition of claim 1 for the treatment of a disease in a subject. 53. The composition of claim 52 , wherein the disease is a proliferative disease. 54. The composition of claim 53 , wherein the proliferative disease is cancer. 54. The composition of claim 53 , wherein the proliferative disease is psoriasis. 53. The composition of claim 52 , wherein the disease is hypertension. 53. The composition of claim 52 , wherein the disease is obesity. 53. The composition of claim 52 , wherein the disease is diabetes. 53. The composition according to claim 52 , wherein the disease is a central nervous system disease or a neurodegenerative disease. 53. The composition of claim 52 , wherein the disease is pain. 53. The composition of claim 52 , wherein the disease is Alzheimer's disease, amyotrophic lateral sclerosis, dementia or Parkinson's disease. 53. The composition of claim 52 , wherein the disease is stroke. 53. The composition of claim 52 , wherein the disease is an inflammatory disease. Wherein the inflammatory disease is rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, according to claim 63 which is selected from the group consisting of atherosclerosis, chronic obstructive pulmonary disease (COPD) and multiple sclerosis A composition according to 1. 53. The composition of claim 52 , wherein the disease is premalignant neoplasia or dysplasia. 66. The composition of claim 65 , wherein the disease is intraepithelial neoplasia. 53. The composition of claim 52 , wherein the disease is an infectious disease. 68. The composition of claim 67 , wherein the infection is a viral infection. 69. The composition of claim 68 , wherein the virus is selected from the group consisting of HIV, HTLV, HPV, HSV, HBV, EBV, varicella-zoster virus, adenovirus, parvovirus or JC virus.   A kit for the treatment of a disease comprising the composition of claim 1 and instructions for its use.

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